Glypican-3-binding fibronectin based scaffold molecules

ABSTRACT

Provided herein are polypeptides which include tenth fibronectin type III domains (10Fn3) that bind to glypican-3. Also provided are fusion molecules comprising a 10Fn3 domain that bind to glypican-3 for use in diagnostic and therapeutic applications. Glypican-3 10Fn3 drug conjugates are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/760,449, filed on Mar. 15, 2018, which is a 35 U.S.C. 371 nationalstage filing of International Application No. PCT/US2016/053185, filedSep. 22, 2016, and which claims priority to U.S. Provisional ApplicationNo. 62/222,633, filed Sep. 23, 2015. The contents of the aforementionedapplications are hereby incorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 24, 2020,named 2020_01_23_Sequence_Listing_for filing_-_MXI-546USCN.txt and is411,808 bytes in size.

BACKGROUND

Glypican-3 is an oncofetal antigen that belongs to the glypican familyof glycosyl-phosphatidylinositol-anchored heparin sulfate proteoglycans.Glypicans regulate the activity of several growth factors includingWnts, Hedgehogs, bone morphogenic proteins and fibroblast growth factors(FGFs) (Filmus et al. FEBS J. 2013, 280:2471-2476). Glypicans arecharacterized by a covalent linkage to complex polysaccharide chainscalled heparinsulphate glycosaminoglycans. Glypicans are involved incell signaling at the cellular-extracellular matrix interface.(Sasisekharan et al., Nature Reviews|Cancer, Volume 2 (2002). To date,six distinct members of the human glypican family have been identified.Cell membrane-bound glypican-3 is composed of two subunits, linked byone or more disulfide bonds.

Glypican-3 is expressed in fetal liver and placenta during developmentand is down-regulated or silenced in normal adult tissues. Mutations anddepletions in the glypican-3 gene are responsible for theSimpson-Golabi-Behmel or Simpson dysmorphia syndrome in humans.Glypican-3 is expressed in various cancers and, in particular,hepatocellular carcinoma (“HCC”), melanoma, Wilm's tumor, andhepatoblastoma. (Jakubovic and Jothy; Ex. Mol. Path. 82:184-189 (2007);Nakatsura and Nishimura, Biodrugs 19(2):71-77 (2005). The cell surfaceform of Glypican-3 is highly expressed in HCC (>50%) and in othercancers including squamous lung cancer (approximately 25%).

Glypican-3 promotes tumor growth in vitro and in vivo by stimulatingcanonical Wnt signaling which induces the cytosolic accumulation andnuclear translocation of the transcription factor β-catenin (Filmus,supra). It has been shown that GPC3 can form a complex with several Wnts(Capurro et al., Cancer Res., 2005, 65:6245-6254), and Frizzleds, thesignaling receptor for Wnts (Filmus, et al., Genome Biol., 2008, 9:224).

HCC is the third leading cause of cancer-related deaths worldwide. Eachyear, HCC accounts for about 1 million deaths. (Nakatsura and Nishimura,Biodrugs 19(2):71-77 (2005).) Hepatitis B virus, hepatitis C virus, andchronic heavy alcohol use leading to cirrhosis of the liver remain themost common causes of HCC. Its incidence has increased dramatically inthe United States because of the spread of hepatitis C virus infectionand is expected to increase for the next 2 decades. HCC is treatedprimarily by liver transplantation or tumor resection. Patient prognosisis dependent on both the underlying liver function and the stage atwhich the tumor is diagnosed. (Parikh and Hyman, Am J. Med.120(3):194-202 (2007).) Effective HCC treatment strategies are needed.

SUMMARY

Provided herein are polypeptides containing fibronectin based scaffolds(FBS) that bind to human glypican-3 and conjugates of these polypeptidesthat are suitable as therapeutic and diagnostic agents.

In one aspect, provided is a polypeptide comprising an FBS whichspecifically binds to human glypican-3 (GPC3). In some embodiments, theanti-GPC3 FBS is conjugated to therapeutic or diagnostic agent.

In another aspect, provided are methods of treating cancer in a humansubject by administering to the subject a therapeutically effectiveamount of a polypeptide comprising an anti-GPC3 FBS or an anti-GPC3FBS-drug conjugate. In some embodiments, the cancer overexpressesglypican-3 relative to non-cancerous cells. In some embodiments, thecancer is liver cancer (e.g., hepatocelluar carncinoma, hepatoblastoma),melanoma, Wilm's tumor or lung cancer (e.g., squamous lung cancer).

In another aspect, provided are methods of detecting GPC3 in vitro andin vivo, comprising contacting a cell with a polypeptide comprising ananti-GPC3 FBS under conditions to allow binding of the anti-GPC3 FBS toGPC3, and detecting complexes comprising the anti-GPC3 FBS and GPC3. Insome embodiments, the anti-GPC3 FBS is linked to a detectable label(e.g., FITC).

Also provided are compositions, including pharmaceutical and diagnosticcompositions, comprising the anti-GPC3 FBS polypeptides and/or anti-GPC3FBS drug conjugates.

Also provided are nucleic acid molecules encoding the anti-GPC3 FBS, aswell as expression vectors comprising such nucleic acids and host cellscomprising such expression vectors. Also provided are kits comprisingthe anti-GPC3 FBS polypeptides and anti-GPC3 FBS conjugates andinstructions for use.

Other features and advantages of the instant invention will be apparentfrom the following detailed description and examples which should not beconstrued as limiting.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the amino acid sequences of representative anti-GPC3adnectin polypeptides. 4578F03 (SEQ ID NO: 9), 4578H08 (SEQ ID NO: 18),4578B06 (SEQ ID NO: 35), 4606F06 (SEQ ID NO: 48), 5273C01 (SEQ ID NO:61), 5273D01 (SEQ ID NO: 74), 5274E01 (SEQ ID NO: 87), 6561A01 (SEQ IDNO: 87), 6077F02 (SEQ ID NO: 102), 6093A01 (SEQ ID NO: 463).

FIGS. 2A and 2B are schematics depicting the structure of the DAR1 andDAR2 tubulysin analog-GPC3 Adnectin drug conjugates.

FIGS. 3A-3D show flow cytometry results of anti-GPC3 Adnectins bindingto human Glypican-3 positive cells.

FIGS. 4A and 4B show flow cytometry results of anti-GPC3 AdxDC DAR1 andDAR2 binding to human Hep3B and H446 cells.

FIGS. 5A and 5B show cell growth inhibition of Hep3B and H446 cells byanti-GPC3 AdxDC DAR1 and DAR2.

FIGS. 6A and 6B show cell growth inhibition of Hep3B and HepG2 cells byanti-GPC3 AdxDC DAR1 and DAR2.

FIGS. 7A and 7B are bar graphs depicting the internalization rate of theanti-GPC3 adnectin, ADX_6077_F02, into H446 and HepB3 cells

FIGS. 8A-8E depict the membrane and internalized AF488 labeled anti-GPC3adnectin, ADX_6077_F02, at the 15 minute and 8 hour time points.

FIG. 9 is a graph depicting the exposure profile of the tubulysinanalog-anti-GPC3 adnectin drug conjugate in mice.

FIGS. 10A and 10B are graphs depicting the efficacy of anti-GPC3adnectin drug conjugates in a HepG2 xenograft model, as measured bytumor volume shrinkage and percent body weight change.

FIGS. 11A-11D are graphs depicting the efficacy of DAR1 and DAR2 in aHepG2 xenograft model (TV₀=380-480 mm³), as measured by tumor volumeshrinkage and percent body weight change.

FIGS. 12A and 12B are graphs depicting the efficacy of DAR2 in a HepG2xenograft model (TV₀=228-350 mm³), as measured by tumor volume shrinkageand percent body weight change.

FIGS. 13A and 13B are graphs depicting the efficacy of DAR2 in a HepG2xenograft model (TV₀=514-673 mm³), as measured by tumor volume shrinkageand percent body weight change.

FIG. 14 depicts the common peptic peptides for human GPC3 (amino acids1-559 of SEQ ID NO: 344, followed by 6×his) determined by massspectrometry.

FIG. 15 is a graphic depiction of the ADX_6077_F02 adnectin binding siteon human GPC3, as determined by HDX-MS.

FIGS. 16A and 16B are graphic comparisons of the binding kinetics ofanti-GPC3 DG mutants with the parent anti-GPC3 adnectin.

FIG. 17 shows tumor volume as a function of days after administration ofvarious doses of GPC3_AdxDC DA or control non-binding AdxDC to NSG miceimplanted with Hep3B tumor cells, showing that GPC3_AdxDC DA isefficacious in cell line derived xenografts with high expression ofglypican-3.

FIG. 18 shows tumor volume as a function of days after administration ofvarious doses of GPC3_AdxDC DA or control non binding AdxDC to CB17 SCIDmice implanted with H446 tumor cells, showing that GPC3_AdxDC DA slowsgrowth of cell line derived xenografts with low expression ofglypican-3.

FIG. 19 shows Quantitative Whole-Body Autoradiography (QWBA) of micetissues taken 0.17 hours, 1 hour, 5 hours and 168 hours afteradministration of 0.22 M/kg of ³H GPC3_AdxDC to the mice, showingpreferential uptake to Hep3B tumor relative to normal tissues.

FIG. 20 shows QWBA of mice tissues taken 0.17 hours, 1 hour, 5 hours and168 hours after administration of 0.015 μM/kg of ³H GPC3_AdxDC to themice, showing preferential uptake to Hep3B tumor relative to normaltissues.

FIG. 21 shows QWBA of mice tissues taken 0.17 hours, 1 hour, 5 hours and168 hours after administration of 0.22 μM/kg of ³H GPC3_AdxDC to themice, showing a higher uptake to Hep3B tumor relative to that ofnon-binding control AdxDC.

FIG. 22 shows QWBA of mice tissues taken 0.17 hours, 1 hour, 5 hours and168 hours after administration of 0.22 M/kg of ³H RGE_AdxDC (control,non GPC3-binding AdxDC) to the mice, showing a lower uptake to Hep3Btumor relative to that of GPC3_AdxDC.

FIG. 23 shows the total radioactivity concentration in various mousetissues at 0.17 hours, 1 hour, 5 hours, 24 hours, 48 hours and 168 hoursafter administration of ³H GPC3_AdxDC or non-binding AdxDC control(“RGE_ADxDC”) administered to mice. The bars for each tissue are shownin the order set forth in the previous sentence.

FIGS. 24A and 24B show heat maps of the BC loop of GPC3_AdxDC DG (aminoacids 15-21 of SEQ ID NO: 98) obtained by positional scanning using 10nM (FIG. 24A) or 1 nM (FIG. 24B) human glypican-3-biotin. The sequenceof the wt BC loop is set forth in amino acids 15-21 of SEQ ID NO: 1.

FIGS. 25A and 25B show heat maps of the BC loop of GPC3_AdxDC DA (aminoacids 15-21 of SEQ ID NO: 98) obtained by positional scanning using 10nM (FIG. 25A) or 1 nM (FIG. 25B) human glypican-3-biotin. The sequenceof the wt BC loop is set forth in amino acids 15-21 of SEQ ID NO: 1.

FIGS. 26A and 26B show heat maps of the DE loop of GPC3_AdxDC DGobtained by positional scanning using 10 nM (FIG. 26A) or 1 nM (FIG.26B) human glypican-3-biotin. The sequence of the wt DE loop is setforth in amino acids 52-55 SEQ ID NO: 1.

FIGS. 27A and 27B show heat maps of the DE loop of GPC3_AdxDC DAobtained by positional scanning using 10 nM (FIG. 27A) or 1 nM (FIG.27B) human glypican-3-biotin. The sequence of the wt DE loop is setforth in amino acids 52-55 of SEQ ID NO: 1.

FIG. 28 shows heat maps of the FG loop of GPC3_AdxDC DG (amino acids69-79 of SEQ ID NO: 98) obtained by positional scanning using 10 nMhuman glypican-3-biotin. The sequence of the wt FG loop is set forth inamino acids 77-87 SEQ ID NO: 1.

FIG. 29 shows heat maps of the FG loop of GPC3_AdxDC DG (amino acids69-79 of SEQ ID NO: 98) obtained by positional scanning using 1 nM humanglypican-3-biotin. The sequence of the wt FG loop is set forth in aminoacids 77-87 SEQ ID NO: 1.

FIG. 30 shows heat maps of the FG loop of GPC3_AdxDC DA (amino acids69-79 of SEQ ID NO: 98) obtained by positional scanning using 10 nMhuman glypican-3-biotin. The sequence of the wt FG loop is set forth inamino acids 77-87 SEQ ID NO: 1.

FIG. 31 shows heat maps of the FG loop of GPC3_AdxDC DA (amino acids69-79 of SEQ ID NO: 98) obtained by positional scanning using 1 nM humanglypican-3-biotin. The sequence of the wt FG loop is set forth in aminoacids 77-87 SEQ ID NO: 1.

DETAILED DESCRIPTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by the skilled artisan.Although any methods and compositions similar or equivalent to thosedescribed herein can be used in practice or testing of the presentinvention, the preferred methods and compositions are described herein.

The terms “glypican-3, “glypican proteoglycan 3,” “GPC3,”“OTTHUMP00000062492,” “GTR2-2,” “SGB,” “DGSX,” “SDYS,” “SGBS,” “OCI-5,”and “SGBS1” are used interchangeably, and include variants, isoforms andspecies homologs of human glypican-3. The complete amino acid sequenceof an exemplary human glypican-3 has Genbank/NCBI accession numberNM_004484 (SEQ ID NO: 344).

An “amino acid residue” is the remaining portion of an amino acid aftera water molecule has been lost (an H+ from the nitrogenous side and anOH— from the carboxylic side) in the formation of a peptide bond.

By “polypeptide” is meant any sequence of two or more amino acids,regardless of length, post-translation modification, or function.Polypeptides can include natural amino acids and non-natural amino acidssuch as those described in U.S. Pat. No. 6,559,126, incorporated hereinby reference. Polypeptides can also be modified in any of a variety ofstandard chemical ways (e.g., an amino acid can be modified with aprotecting group; the carboxy-terminal amino acid can be made into aterminal amide group; the amino-terminal residue can be modified withgroups to, e.g., enhance lipophilicity; or the polypeptide can bechemically glycosylated or otherwise modified to increase stability orin vivo half-life). Polypeptide modifications can include the attachmentof another structure such as a cyclic compound or other molecule to thepolypeptide and can also include polypeptides that contain one or moreamino acids in an altered configuration (i.e., R or S; or, L or D).

An “isolated” polypeptide is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the polypeptide,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In certain embodiments, the polypeptide willbe purified (1) to greater than 95% by weight of polypeptide asdetermined by the Lowry method, and most preferably more than 99% byweight, (2) to a degree sufficient to obtain at least residues ofN-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (3) to homogeneity by SDS-PAGE under reducing ornonreducing condition using Coomassie blue or, preferably, silver stain.

As used herein, a “¹⁰Fn3 domain” or “¹⁰Fn3 moiety” or “¹⁰Fn3 molecule”refers to wild-type ¹⁰Fn3 and biologically active variants thereof,e.g., biologically active variants that specifically bind to a target,such as a target protein. A wild-type human ¹⁰Fn3 domain may comprisethe amino acid sequence set forth in SEQ ID NO: 1. Biologically activevariants of a wild-type human ¹⁰Fn3 domain include ¹⁰Fn3 domains thatcomprise at least, at most or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40 or 45amino acid changes, i.e., substitutions, additions or deletions,relative to a ¹⁰Fn3 domain comprising SEQ ID NOs: 1.

An “Adnectin” or “Adx” or “adnectin” or “adx” refers to a human ¹⁰Fn3molecule that has been modified (relative to the wild-type sequence) tobind specifically to a target.

A “GPC3 Adnectin” or “anti-GPC3 Adnectin” is an Adnectin that bindsspecifically to GPC3, e.g., with a K_(D) of 1 μM or less.

A “region” of a ¹⁰Fn3 domain (or moiety or molecule) as used hereinrefers to either a loop (AB, BC, CD, DE, EF and FG), a β-strand (A, B,C, D, E, F and G), the N-terminus (corresponding to amino acid residues1-7 of SEQ ID NO: 1), or the C-terminus (corresponding to amino acidresidues 93-94 of SEQ ID NO: 1).

A “north pole loop” of a ¹⁰Fn3 domain (or moiety) refers to any one ofthe BC, DE and FG loops of a ¹⁰Fn3 domain.

A “south pole loop” of a ¹⁰Fn3 domain (or moiety) refers to any one ofthe AB, CD and EF loops of a ¹⁰Fn3 domain.

A “scaffold region” refers to any non-loop region of a human ¹⁰Fn3domain. The scaffold region includes the A, B, C, D, E, F and Gβ-strands as well as the N-terminal region (amino acids corresponding toresidues 1-7 of SEQ ID NO: 1) and the C-terminal region (amino acidscorresponding to residues 93-94 of SEQ ID NO: 1).

“Percent (%) amino acid sequence identity” herein is defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in a selected sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST℠,BLAST℠-2, ALIGN, ALIGN-2 or Megalign (DNASTAR®) software. Those skilledin the art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull-length of the sequences being compared.

For purposes herein, the % amino acid sequence identity of a given aminoacid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows: 100times the fraction X/Y where X is the number of amino acid residuesscored as identical matches by a sequence alignment program, such asBLAST℠, BLAST℠-2, ALIGN, ALIGN-2 or Megalign (DNASTAR®), in thatprogram's alignment of A and B, and where Y is the total number of aminoacid residues in B. It will be appreciated that where the length ofamino acid sequence A is not equal to the length of amino acid sequenceB, the % amino acid sequence identity of A to B will not equal the %amino acid sequence identity of B to A.

As used herein, the term “Adnectin binding site” refers to the site orportion of a protein (e.g., GPC3) that interacts or binds to aparticular Adnectin. Adnectin binding sites can be formed fromcontiguous amino acids or noncontiguous amino acids juxtaposed bytertiary folding of a protein. Adnectin binding sites formed bycontiguous amino acids are typically retained on exposure to denaturingsolvents, whereas Adnectin binding sites formed by tertiary folding aretypically lost on treatment of denaturing solvents.

An Adnectin binding site for an anti-GPC3 Adnectin described herein maybe determined by application of standard techniques typically used forepitope mapping of antibodies including, but not limited to proteasemapping and mutational analysis.

As used herein, an amino acid residue in a polypeptide is considered to“contribute to binding” a target if (1) any of the non-hydrogen atoms ofthe residue's side chain or main chain is found to be within fiveangstroms of any atom of the binding target based on an experimentallydetermined three-dimensional structure of the complex, and/or (2)mutation of the residue to its equivalent in wild-type ¹⁰Fn3 (e.g., SEQID NO: 1), to alanine, or to a residue having a similarly sized orsmaller side chain than the residue in question, leads to a measuredincrease of the equilibrium dissociation constant to the target (e.g.,an increase in the k_(on)).

The terms “specifically binds,” “specific binding,” “selective binding,and “selectively binds,” as used interchangeably herein in the contextof FBS binding to GPC3 refers to an FBS that exhibits affinity for GPC3,but does not significantly bind (e.g., less than about 10% binding) to adifferent polypeptides as measured by a technique available in the artsuch as, but not limited to, Scatchard analysis and/or competitivebinding assays (e.g., competition ELISA, BIACORE assay). The term isalso applicable where e.g., a binding domain of an FBS described hereinis specific for GPC3 from one or more species (e.g., human, rodent,primate), but does not does not cross-react with other glypicans (e.g.,glypican-1, glypican-2, glypican-5, glypican-6).

The term “preferentially binds” as used herein in the context ofAdnectins binding to GPC3 refers to the situation in which an Adnectindescribed herein binds GPC3 at least about 20% greater than it binds adifferent polypeptide as measured by a technique available in the artsuch as, but not limited to, Scatchard analysis and/or competitivebinding assays (e.g., competition ELISA, BIACORE assay).

The term “K_(D),” as used herein, e.g., in the context of Adnectinsbinding to GPC3, is intended to refer to the dissociation equilibriumconstant of a particular Adnectin-protein (e.g., GPC3) interaction orthe affinity of an Adnectin for a protein (e.g., GPC3), as measuredusing a surface plasmon resonance assay or a cell binding assay. A“desired K_(D),” as used herein, refers to a K_(D) of an Adnectin thatis sufficient for the purposes contemplated. For example, a desiredK_(D) may refer to the K_(D) of an Adnectin required to elicit afunctional effect in an in vitro assay, e.g., a cell-based luciferaseassay.

The term “k_(a)”, as used herein in the context of Adnectins binding toa protein, is intended to refer to the association rate constant for theassociation of an Adnectin into the Adnectin/protein complex.

The term “k_(d)”, as used herein in the context of Adnectins binding toa protein, is intended to refer to the dissociation rate constant forthe dissociation of an Adnectin from the Adnectin/protein complex.

The term “IC₅₀”, as used herein in the context of Adnectins, refers tothe concentration of an Adnectin that inhibits a response, either in anin vitro or an in vivo assay, to a level that is 50% of the maximalinhibitory response, i.e., halfway between the maximal inhibitoryresponse and the untreated response.

The term “glypican activity” or “glypican-3” activity as used hereinrefers to one or more of growth-regulatory or morphogenetic activitiesassociated with activation of cell signaling by GPC3, for example,activation of Wnt signaling. For example, GPC3 may modulate tumor cellgrowth by complex formation with Wnt and/or Frizzeled proteins. GPC3 mayalso activate signaling pathways and tumor cell growth by interactingwith FGF. GPC3 activity can be determined using art-recognized methods,such as those described herein. The phrases “glypican-3 activity” or“antagonize glypican-3 activity” or “antagonize glypican-3” are usedinterchangeably to refer to the ability of the anti-GPC3 FBS andanti-GPC3 drug conjugates provided herein to neutralize or antagonize anactivity of GPC3 in vivo or in vitro. The terms “inhibit” or“neutralize” as used herein with respect to an activity of an anti-GPC3FBS means the ability to substantially antagonize, prohibit, prevent,restrain, slow, disrupt, eliminate, stop, reduce or reverse e.g.,progression or severity of that which is being inhibited including, butnot limited to, a biological activity or property, a disease or acondition (e.g., tumor cell growth). The inhibition or neutralization ispreferably at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95% or higher.

As used herein, the term “linked” refers to the association of two ormore molecules. The linkage can be covalent or non-covalent. The linkagemay be between a polypeptide and a chemical moiety or anotherpolypeptide moiety. The linkage also can be genetic (i.e., recombinantlyfused). Such linkages can be achieved using a wide variety of artrecognized techniques, such as chemical conjugation and recombinantprotein production.

The term “PK” is an acronym for “pharmacokinetic” and encompassesproperties of a compound including, by way of example, absorption,distribution, metabolism, and elimination by a subject. A “PK modulationprotein” or “PK moiety” as used herein refers to any protein, peptide,or moiety that affects the pharmacokinetic properties of a biologicallyactive molecule when fused to or administered together with thebiologically active molecule. Examples of a PK modulation protein or PKmoiety include PEG, human serum albumin (HSA) binders (as disclosed inU.S. Publication Nos. 2005/0287153 and 2007/0003549, PCT PublicationNos. WO 2009/083804 and WO 2009/133208), human serum albumin andvariants thereof, transferrin and variants thereof, Fc or Fc fragmentsand variants thereof, and sugars (e.g., sialic acid).

The serum or plasma “half-life” of a polypeptide can generally bedefined as the time taken for the serum concentration of the polypeptideto be reduced by 50%, in vivo, for example due to degradation of thepolypeptide and/or clearance or sequestration of the polypeptide bynatural mechanisms. The half-life can be determined in any manner knownper se, such as by pharmacokinetic analysis. Suitable techniques will beclear to the person skilled in the art, and may, for example, generallyinvolve the steps of administering a suitable dose of a polypeptide to aprimate; collecting blood samples or other samples from said primate atregular intervals; determining the level or concentration of thepolypeptide in said blood sample; and calculating, from (a plot of) thedata thus obtained, the time until the level or concentration of thepolypeptide has been reduced by 50% compared to the initial level upondosing. Methods for determining half-life may be found, for example, inKenneth et al., Chemical Stability of Pharmaceuticals: A Handbook forPharmacists (1986); Peters et al., Pharmacokinete Analysis: A PracticalApproach (1996); and Gibaldi, M. et al., Pharmacokinetics, Second Rev.Edition, Marcel Dekker (1982).

Serum half-life can be expressed using parameters such as thet_(1/2)-alpha, t_(1/2)-beta and the area under the curve (AUC). An“increase in half-life” refers to an increase in any one of theseparameters, any two of these parameters, or in all three theseparameters. In certain embodiments, an increase in half-life refers toan increase in the t_(1/2)-beta and/or HL Lambda z, either with orwithout an increase in the t_(1/2)-alpha and/or the AUC or both.

The term “detectable” refers to the ability to detect a signal over thebackground signal. The term “detectable signal” is a signal derived fromnon-invasive imaging techniques such as, but not limited to, positronemission tomography (PET). The detectable signal is detectable anddistinguishable from other background signals that may be generated fromthe subject. In other words, there is a measurable and statisticallysignificant difference (e.g., a statistically significant difference isenough of a difference to distinguish among the detectable signal andthe background, such as about 0.1%, 1%, 3%, 5%, 10%, 15%, 20%, 25%, 30%,or 40% or more difference between the detectable signal and thebackground) between the detectable signal and the background. Standardsand/or calibration curves can be used to determine the relativeintensity of the detectable signal and/or the background.

A “detectably effective amount” of a composition comprising an imagingagent described herein is defined as an amount sufficient to yield anacceptable image using equipment that is available for clinical use. Adetectably effective amount of an imaging agent provided herein may beadministered in more than one injection. The detectably effective amountcan vary according to factors such as the degree of susceptibility ofthe individual, the age, sex, and weight of the individual,idiosyncratic responses of the individual, and the like. Detectablyeffective amounts of imaging compositions can also vary according toinstrument and methodologies used. Optimization of such factors is wellwithin the level of skill in the art. In certain embodiments, a GPC3imaging agent, e.g., those described herein, provides a differentiationfactor (i.e., specific signal to background signal) of 2 or more, e.g.,3, 4, 5 or more.

The terms “individual,” “subject,” and “patient,” used interchangeablyherein, refer to an animal, preferably a mammal (including a nonprimateand a primate), e.g., a human.

A “cancer” refers a broad group of various diseases characterized by theuncontrolled growth of abnormal cells in the body. Unregulated celldivision and growth divide and grow results in the formation ofmalignant tumors that invade neighboring tissues and may alsometastasize to distant parts of the body through the lymphatic system orbloodstream.

“Treatment” or “therapy” of a subject refers to any type of interventionor process performed on, or the administration of an active agent to,the subject with the objective of reversing, alleviating, ameliorating,inhibiting, slowing down or preventing the onset, progression,development, severity or recurrence of a symptom, complication,condition or biochemical indicia associated with a disease.

“Administration” or “administering,” as used herein in the context ofanti-GPC3 Adnectins, refers to introducing a GPC3 Adnectin or GPC3Adnectin-based probe or a labeled probe (also referred to as the“imaging agent”) described herein into a subject. Any route ofadministration is suitable, such as intravenous, oral, topical,subcutaneous, peritoneal, intra-arterial, inhalation, vaginal, rectal,nasal, introduction into the cerebrospinal fluid, or instillation intobody compartments can be used.

The terms “co-administration” or the like, as used herein, are meant toencompass administration of the selected pharmaceutical agents to asingle patient, and are intended to include regimens in which the agentsare administered by the same or different route of administration or atthe same or different time.

The term “therapeutically effective amount” refers to at least theminimal dose, but less than a toxic dose, of an agent which is necessaryto impart a therapeutic benefit to a subject.

As used herein, an “effective amount” refers to at least an amounteffective, at dosages and for periods of time necessary, to achieve thedesired result.

As used herein, a “sufficient amount” refers to an amount sufficient toachieve the desired result.

The term “sample” can refer to a tissue sample, cell sample, a fluidsample, and the like. The sample may be taken from a subject. The tissuesample can include hair (including roots), buccal swabs, blood, saliva,semen, muscle, or from any internal organs. The fluid may be, but is notlimited to, urine, blood, ascites, pleural fluid, spinal fluid, and thelike. The body tissue can include, but is not limited to, skin, muscle,endometrial, uterine, and cervical tissue.

Overview

Provided herein are novel fibronectin based scaffold polypeptides whichbind to human glypican-3. Such polypeptides can be coupled to othertherapeutic and diagnostic agents and are useful, for example, intargeting therapeutic and diagnostic agents to cells and tissuesexpressing glypican-3 (e.g., cancer cells over-expressing glypican-3).

I. Anti-GPC3 Fibronectin Based Scaffolds

As used herein, a “fibronectin based scaffold” or “FBS” protein ormoiety refers to proteins or moieties that are based on a fibronectintype III (“Fn3”) repeat and can be modified to bind specifically togiven targets, e.g., target proteins. Fn3 is a small (about 10 kDa)domain that has the structure of an immunoglobulin (Ig) fold (i.e., anIg-like β-sandwich structure, consisting of seven β-strands and sixloops). Fibronectin has 18 Fn3 repeats, and while the sequence homologybetween the repeats is low, they all share a high similarity in tertiarystructure. Fn3 domains are also present in many proteins other thanfibronectin, such as adhesion molecules, cell surface molecules, e.g.,cytokine receptors, and carbohydrate binding domains. For reviews seeBork et al., Proc. Natl. Acad. Sci. USA, 89(19):8990-8994 (1992); Borket al., J. Mol. Biol., 242(4):309-320 (1994); Campbell et al.,Structure, 2(5):333-337 (1994); Harpez et al., J. Mol. Biol.,238(4):528-539 (1994)). The term “FBS” protein or moiety is intended toinclude scaffolds based on Fn3 domains from these other proteins (i.e.,non fibronectin molecules).

An Fn3 domain is small, monomeric, soluble, and stable. It lacksdisulfide bonds and, therefore, is stable under reducing conditions. Fn3domains comprise, in order from N-terminus to C-terminus, a beta orbeta-like strand, A; a loop, AB; a beta or beta-like strand, B; a loop,BC; a beta or beta-like strand, C; a loop, CD; a beta or beta-likestrand, D; a loop, DE; a beta or beta-like strand, E; a loop, EF; a betaor beta-like strand, F; a loop, FG; and a beta or beta-like strand, G.The seven antiparallel β-strands are arranged as two beta sheets thatform a stable core, while creating two “faces” composed of the loopsthat connect the beta or beta-like strands. Loops AB, CD, and EF arelocated at one face (“the south pole”) and loops BC, DE, and FG arelocated on the opposing face (“the north pole”).

The loops in Fn3 molecules are structurally similar to complementarydetermining regions (CDRs) of antibodies, and when altered, may beinvolved in binding of the Fn3 molecule to a target, e.g., a targetprotein. Other regions of Fn3 molecules, such as the beta or beta-likestrands and N-terminal or C-terminal regions, when altered, may also beinvolved in binding to a target. Any or all of loops AB, BC, CD, DE, EFand FG may participate in binding to a target. Any of the beta orbeta-like strands may be involved in binding to a target. Fn3 domainsmay also bind to a target through one or more loops and one or more betaor beta-like strands. Binding may also require the N-terminal orC-terminal regions.

An anti-GPC3 FBS may be based on the tenth fibronectin type III domain,i.e., the tenth module of human Fn3 (¹⁰Fn3) in which one or more solventaccessible loops have been randomized or mutated. The amino acidsequence of a wild-type human ¹⁰Fn3 moiety is as follows:

(SEQ ID NO: 1) VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT 

The AB, CD and EF loops are underlined; the BC, FG, and DE loops areemphasized in bold; the β-strands are located between or adjacent toeach of the loop regions; and the N-terminal region is shown initalics). The last two amino acid residues of SEQ ID NO: 1 are a portionof a C-terminal region. The core ¹⁰Fn3 domain begins with amino acid 9(“E”) and ends with amino acid 94 (“T”) and corresponds to an 86 aminoacid polypeptide. The core wild-type human ¹Fn3 domain is set forth inSEQ ID NO: 2. Both variant and wild-type ¹⁰Fn3 proteins arecharacterized by the same structure, namely seven beta-strand domainsequences designated A through G and six loop regions (AB loop, BC loop,CD loop, DE loop, EF loop, and FG loop) which connect the sevenbeta-strand domain sequences. The beta strands positioned closest to theN- and C-termini may adopt a beta-like conformation in solution. In SEQID NO: 1, the AB loop corresponds to residues 14-17, the BC loopcorresponds to residues 23-31, the CD loop corresponds to residues37-47, the DE loop corresponds to residues 51-56, the EF loopcorresponds to residues 63-67, and the FG loop corresponds to residues76-87.

Accordingly, in certain embodiments, the anti-GPC3 FBS moiety (e.g.,anti-GPC3 Adnectin) comprises a ¹⁰Fn3 domain that is defined generallyby the following degenerate sequence:

(SEQ ID NO: 3) VSDVPRD LEVVAA (X)_(u) LLISW (X)_(v) YRITY (X)_(w) FTV(X)_(x) ATISGL (X)_(y) YTITVYA (X)_(z) ISINY RT,or by a sequence lacking 1, 2, 3, 4, 5, 6 or 7 N-terminal amino acids,respectively.

In SEQ ID NO: 3, the AB loop is represented by (X)_(u), the BC loop isrepresented by (X)_(v), the CD loop is represented by (X)_(w), the DEloop is represented by (X)_(x), the EF loop is represented by (X)_(y)and the FG loop is represented by X_(z). X represents any amino acid andthe subscript following the X represents an integer of the number ofamino acids. In particular, u, v, w, x, y and z may each independentlybe anywhere from 2-20, 2-15, 2-10, 2-8, 5-20, 5-15, 5-10, 5-8, 6-20,6-15, 6-10, 6-8, 2-7, 5-7, or 6-7 amino acids. The sequences of the betastrands (underlined in SEQ ID NO: 3) may have anywhere from 0 to 10,from 0 to 8, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3, from 0to 2, or from 0 to 1 substitutions, deletions or additions across all 7scaffold regions relative to the corresponding amino acids shown in SEQID NO: 3. In some embodiments, the sequences of the beta strands mayhave anywhere from 0 to 10, from 0 to 8, from 0 to 6, from 0 to 5, from0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1 substitutions, e.g.,conservative substitutions, across all 7 scaffold regions relative tothe corresponding amino acids shown in SEQ ID NO: 3.

It should be understood that not every residue within a loop regionneeds to be modified or altered in order to achieve a ¹Fn3 bindingdomain having strong affinity for a desired target. Additionally,insertions and deletions in the loop regions may also be made whilestill producing high affinity anti-GPC3 ¹⁰Fn3 binding domains. By“altered” is meant one or more amino acid sequence alterations relativeto a template sequence (i.e., the corresponding wild-type humanfibronectin domain) and includes amino acid additions, deletions,substitutions or a combination thereof.

In some embodiments, the anti-GPC3 FBS moiety comprises a ¹⁰Fn3 domainwherein the non-loop regions comprise an amino acid sequence that is atleast 80, 85, 90, 95, 98, or 100% identical to the non-loop regions ofSEQ ID NO: 1, and wherein at least one loop selected from AB, BC, CD,DE, EF and FG is altered.

In some embodiments, one or more loops selected from AB, BC, CD, DE, EFand FG may be extended or shortened in length relative to thecorresponding loop in wild-type human ¹⁰Fn3. In any given polypeptide,one or more loops may be extended in length, one or more loops may bereduced in length, or combinations thereof. In some embodiments, thelength of a given loop may be extended by 2-25, 2-20, 2-15, 2-10, 2-5,5-25, 5-20, 5-15, 5-10, 10-25, 10-20, or 10-15 amino acids. In someembodiments, the length of a given loop may be reduced by 1-15, 1-11,1-10, 1-5, 1-3, 1-2, 2-10, or 2-5 amino acids. In particular, the FGloop of ¹⁰Fn3 is 13 residues long, whereas the corresponding loop inantibody heavy chains ranges from 4-28 residues. Therefore, in someembodiments, the length of the FG loop of ¹⁰Fn3 may be altered in lengthas well as in sequence to obtain the greatest possible flexibility andtarget affinity in polypeptides relying on the FG for target binding.

In certain embodiments, the anti-GPC3 FBS moiety comprises a tenthfibronectin type III (¹⁰Fn3) domain, wherein the ¹⁰Fn3 domain comprisesa loop, AB; a loop, BC; a loop, CD; a loop, DE; a loop EF; and a loopFG; and has at least one loop selected from loop BC, DE, and FG with analtered amino acid sequence relative to the sequence of thecorresponding loop of the human ¹⁰Fn3 domain. In some embodiments, theanti-GPC3 Adnectin described herein comprise a ¹⁰Fn3 domain comprisingan amino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to the non-loop regions of SEQ ID NO: 1 or 2, wherein at leastone loop selected from BC, DE, and FG is altered. In certainembodiments, the BC and FG loops are altered, in certain embodiments,the BC and DE loops are altered, in certain embodiments, the DE and FGloops are altered, and in certain embodiments, the BC, DE, and FG loopsare altered, i.e., the ¹⁰Fn3 domains comprise non-naturally occurringloops. In certain embodiments, the AB, CD and/or the EF loops arealtered. In some embodiments, one or more specific scaffold alterationsare combined with one or more loop alterations.

In some embodiments, the non-ligand binding sequences of the anti-GPC3¹⁰Fn3 may be altered provided that the ¹⁰Fn3 retains ligand bindingfunction and/or structural stability. In some embodiments, the non-loopregion of a ¹Fn3 domain may be modified by one or more conservativesubstitutions. As many as 5%, 10%, 20% or even 30% or more of the aminoacids in the ¹⁰Fn3, domain may be altered by a conservative substitutionwithout substantially altering the affinity of the ¹Fn3 for a ligand. Incertain embodiments, the non-loop regions, e.g., the 3-strands maycomprise anywhere from 0-15, 0-10, 0-8, 0-6, 0-5, 0-4, 0-3, 1-15, 1-10,1-8, 1-6, 1-5, 1-4, 1-3, 2-15, 2-10, 2-8, 2-6, 2-5, 2-4, 5-15, or 5-10conservative amino acid substitutions. In exemplary embodiments, thescaffold modification may reduce the binding affinity of the ¹Fn3 binderfor a ligand by less than 100-fold, 50-fold, 25-fold, 10-fold, 5-fold,or 2-fold. It may be that such changes may alter the immunogenicity ofthe ¹Fn3 in vivo, and where the immunogenicity is decreased, suchchanges may be desirable. As used herein, “conservative substitutions”are residues that are physically or functionally similar to thecorresponding reference residues. That is, a conservative substitutionand its reference residue have similar size, shape, electric charge,chemical properties including the ability to form covalent or hydrogenbonds, or the like. Exemplary conservative substitutions include thosefulfilling the criteria defined for an accepted point mutation inDayhoff et al., Atlas of Protein Sequence and Structure, 5:345-352 (1978and Supp.). Examples of conservative substitutions include substitutionswithin the following groups: (a) valine, glycine; (b) glycine, alanine;(c) valine, isoleucine, leucine; (d) aspartic acid, glutamic acid; (e)asparagine, glutamine; (f) serine, threonine; (g) lysine, arginine,methionine; and (h) phenylalanine, tyrosine.

In some embodiments, one or more of Asp 7, Glu 9, and Asp 23 is replacedby another amino acid, such as, for example, a non-negatively chargedamino acid residue (e.g., Asn, Lys, etc.). A variety of additionalalterations in the ¹Fn3 scaffold that are either beneficial or neutralhave been disclosed. See, for example, Batori et al., Protein Eng.,15(12):1015-1020 (December 2002); Koide et al., Biochemistry,40(34):10326-10333 (Aug. 28, 2001).

In other embodiments, the hydrophobic core amino acid residues (boldedresidues in SEQ ID NO: 3 above) are fixed, and any substitutions,conservative substitutions, deletions or additions occur at residuesother than the hydrophobic core amino acid residues in the ¹⁰Fn3scaffold. Thus, in some embodiments, the hydrophobic core residues ofthe polypeptides provided herein have not been modified relative to thewild-type human ¹⁰Fn3 domain (e.g., SEQ ID NO: 1).

A ¹⁰Fn3 molecule may comprise the flexible linker between the 10th and11^(th) repeat of the Fn3 domain, i.e., EIDKPSQ (SEQ ID NO: 369). Thewild type ¹⁰Fn3 polypeptide with EIDKPSQ (SEQ ID NO: 369) at itsC-terminus is represented by

(SEQ ID NO: 4) VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT EIDKPSQ

In some embodiments, one or more residues of the integrin-binding motif“arginine-glycine-aspartic acid” (RGD) (amino acids 78-80 of SEQ IDNO: 1) may be substituted so as to disrupt integrin binding. In someembodiments, the FG loop of the polypeptides provided herein does notcontain an RGD integrin binding site. In one embodiment, the RGDsequence is replaced by a polar amino acid-neutral amino acid-acidicamino acid sequence (in the N-terminal to C-terminal direction). Incertain embodiments, the RGD sequence is replaced with SGE or RGE.

A. Exemplary Anti-GPC3 Adnectins

In some embodiments, the BC loop of the anti-GPC3 FBS (e.g., an Adnectinthat binds specifically to human GPC3) comprises an amino acid sequenceset forth in SEQ ID NOs: 6, 19, 32, 45, 58, 71, 84 or 99, wherein,optionally, the BC loop comprises 1, 2 or 3 amino acid substitutions,deletions or insertions relative to the BC loop of SEQ ID NOs: 6, 19,32, 45, 58, 71, 84 or 99.

In some embodiments, the DE loop of the anti-GPC3 FBS comprises an aminoacid sequence set forth in SEQ ID NOs: 7, 20, 33, 46, 59, 72, 85 or 100,wherein, optionally, the DE loop comprises 1, 2 or 3 amino acidsubstitutions, deletions or insertions relative to the DE loop of SEQ IDNOs: 7, 20, 33, 46, 59, 72, 85 or 100.

In some embodiments, the FG loop of the anti-GPC3 FBS comprises an aminoacid sequence set forth in SEQ ID NOs: 8, 21, 34, 47, 60, 73, 86, 101,129, 156, 183, 210, 237, 264, 291 or 318, wherein, optionally, the FGloop comprises 1, 2 or 3 amino acid substitutions, deletions orinsertions relative to the FG loop of SEQ ID NOs: 8, 21, 34, 47, 60, 73,86, 101, 129, 156, 183, 210, 237, 264, 291 or 318.

In some embodiments, the BC loop of the anti-GPC3 Adnectin (i.e., anAdnectin that binds specifically to human GPC3) comprises an amino acidsequence set forth in SEQ ID NOs: 6, 19, 32, 45, 58, 71, 84 or 99,wherein, optionally, the BC loop comprises 1, 2 or 3 amino acidsubstitutions, deletions or insertions relative to the BC loop of SEQ IDNOs: 6, 19, 32, 45, 58, 71, 84 or 99; the DE loop of the anti-GPC3Adnectin comprises an amino acid sequence set forth in SEQ ID NOs: 7,20, 33, 46, 59, 72, 85 or 100, wherein, optionally, the DE loopcomprises 1, 2 or 3 amino acid substitutions, deletions or insertionsrelative to the DE loop of SEQ ID NOs: 7, 20, 33, 46, 59, 72, 85 or 100;and the FG loop of the anti-GPC3 FBS comprises an amino acid sequenceset forth in SEQ ID NOs: 8, 21, 34, 47, 60, 73, 86, 101, 129, 156, 183,210, 237, 264, 291 or 318, wherein, optionally, the FG loop comprises 1,2 or 3 amino acid substitutions, deletions or insertions relative to theFG loop of SEQ ID NOs: 8, 21, 34, 47, 60, 73, 86, 101, 129, 156, 183,210, 237, 264, 291 or 318.

In some embodiments, the anti-GPC3 FBS comprises a BC loop comprising anamino acid sequence set forth in SEQ ID NOs: 6, 19, 32, 45, 58, 71, 84or 99; a DE loop comprising amino acid sequence set forth in SEQ ID NOs:7, 20, 33, 46, 59, 72, 85 or 100; and an FG loop comprising an aminoacid sequence set forth in SEQ ID NOs: 8, 21, 34, 47, 60, 73, 86, 101,129, 156, 183, 210, 237, 264, 291 or 318.

In some embodiments, the anti-GPC3 FBS comprises the BC, DE, and FGloops as set forth in SEQ ID NOs: 6, 19, 32, 45, 58, 71, 84 or 99; 7,20, 33, 46, 59, 72, 85 or 100; and 8, 21, 34, 47, 60, 73, 86, 101, 129,156, 183, 210, 237, 264, 291 or 318, respectively, and has amino acidsubstitutions in the BC, DE, and FG loops which allow the FBS tomaintain binding to GPC3.

In some embodiments, the anti-GPC3 FBS comprises the amino acid sequenceset forth in SEQ ID NO: 3, wherein BC, DE and FG loops as represented by(X)_(v), (X)_(x), and (X)_(z), respectively, have amino acid sequencesat least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the BC,DE or FG loop sequences set forth in SEQ ID NOs: 6, 7 and 8,respectively.

In some embodiments, the anti-GPC3 Adnectin comprises the amino acidsequence set forth in SEQ ID NO: 3, wherein the BC, DE, and FG loopscomprise the amino acid sequences set forth in SEQ ID NOs: 6, 7 and 8,respectively, wherein the BC loop has 0, 1, 2, 3, 4, 5, or 6 amino acidsubstitutions, such as conservative amino acid substitutions, and the DEloop has 0, 1, 2 or 3 amino acid substitutions, such as a conservativeamino acid substitution, and the FG loop has 0, 1, 2, 3, 4, 5, 6, 7, or8 amino acid substitutions, such as conservative amino acidsubstitutions.

In certain embodiments, an anti-GPC3 Adnectin (e.g., an anti-FBS moietycomprising a human ¹⁰Fn3) comprises the sequence set forth in SEQ ID NO:3, wherein BC, DE and FG loops as represented by (X)_(v), (X)_(x), and(X)_(z), respectively, comprise BC, DE, and FG loops having the aminoacid sequences of SEQ ID NOs: 6, 7 and 8, respectively.

In certain embodiments, an anti-GPC3 Adnectin comprises the sequence setforth in SEQ ID NO: 3, wherein BC, DE and FG loops as represented by(X)_(v), (X)_(x), and (X)_(z), respectively, have amino acid sequencesat least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the BC,DE or FG loop sequences set forth in SEQ ID NOs: 19, 20 and 21,respectively.

In some embodiments, the anti-GPC3 Adnectin comprises the amino acidsequence set forth in SEQ ID NO: 3, wherein the BC, DE, and FG loopscomprise the amino acid sequences set forth in SEQ ID NOs: 19, 20 and21, respectively, wherein the BC loop has 0, 1, 2, 3, 4, 5, or 6 aminoacid substitutions, such as conservative amino acid substitutions, andthe DE loop has 0, 1, 2 or 3 amino acid substitutions, such as aconservative amino acid substitution, and the FG loop has 0, 1, 2, 3, 4,5, 6, 7, or 8 amino acid substitutions, such as conservative amino acidsubstitutions.

In certain embodiments, an anti-GPC3 Adnectin comprises the sequence setforth in SEQ ID NO: 3, wherein BC, DE and FG loops as represented by(X)_(v), (X)_(x), and (X)_(z), respectively, comprise BC, DE, and FGloops having the amino acid sequences of SEQ ID NOs: 19, 20 and 21,respectively.

In certain embodiments, an anti-GPC3 Adnectin comprises the sequence setforth in SEQ ID NO: 3, wherein BC, DE and FG loops as represented by(X)_(x), (X)_(x), and (X)_(z), respectively, have amino acid sequencesat least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the BC,DE or FG loop sequences set forth in SEQ ID NOs: 32, 33 and 34,respectively.

In some embodiments, the anti-GPC3 Adnectin comprises the amino acidsequence set forth in SEQ ID NO: 3, wherein the BC, DE, and FG loopscomprise the amino acid sequences set forth in SEQ ID NOs: 32, 33 and34, respectively, wherein the BC loop has 0, 1, 2, 3, 4, 5, or 6 aminoacid substitutions, such as conservative amino acid substitutions, andthe DE loop has 0, 1, 2 or 3 amino acid substitutions, such as aconservative amino acid substitution, and the FG loop has 0, 1, 2, 3, 4,5, 6, 7, or 8 amino acid substitutions, such as conservative amino acidsubstitutions.

In certain embodiments, an anti-GPC3 Adnectin comprises the sequence setforth in SEQ ID NO: 3, wherein BC, DE and FG loops as represented by(X)_(v), (X)_(x), and (X)_(z), respectively, comprise BC, DE, and FGloops having the amino acid sequences of SEQ ID NOs: 32, 33 and 34,respectively.

In certain embodiments, an anti-GPC3 Adnectin comprises the sequence setforth in SEQ ID NO: 3, wherein BC, DE and FG loops as represented by(X)_(v), (X)_(x), and (X)_(z), respectively, have amino acid sequencesat least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the BC,DE or FG loop sequences set forth in SEQ ID NOs: 45, 46 and 47,respectively.

In some embodiments, the anti-GPC3 Adnectin comprises the amino acidsequence set forth in SEQ ID NO: 3, wherein the BC, DE, and FG loopscomprise the amino acid sequences set forth in SEQ ID NOs: 45, 46 and47, respectively, wherein the BC loop has 0, 1, 2, 3, 4, 5, or 6 aminoacid substitutions, such as conservative amino acid substitutions, andthe DE loop has 0, 1, 2 or 3 amino acid substitutions, such as aconservative amino acid substitution, and the FG loop has 0, 1, 2, 3, 4,5, 6, 7, or 8 amino acid substitutions, such as conservative amino acidsubstitutions.

In certain embodiments, an anti-GPC3 Adnectin comprises the sequence setforth in SEQ ID NO: 3, wherein BC, DE and FG loops as represented by(X)_(v), (X)_(x), and (X)_(z), respectively, comprise BC, DE, and FGloops having the amino acid sequences of SEQ ID NOs: 45, 46 and 47,respectively.

In certain embodiments, an anti-GPC3 Adnectin comprises the sequence setforth in SEQ ID NO: 3, wherein BC, DE and FG loops as represented by(X)_(v), (X)_(x), and (X)_(z), respectively, have amino acid sequencesat least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the BC,DE or FG loop sequences set forth in SEQ ID NOs: 58, 59 and 60,respectively.

In some embodiments, the anti-GPC3 Adnectin comprises the amino acidsequence set forth in SEQ ID NO: 3, wherein the BC, DE, and FG loopscomprise the amino acid sequences set forth in SEQ ID NOs: 58, 59 and60, respectively, wherein the BC loop has 0, 1, 2, 3, 4, 5, or 6 aminoacid substitutions, such as conservative amino acid substitutions, andthe DE loop has 0, 1, 2 or 3 amino acid substitutions, such as aconservative amino acid substitution, and the FG loop has 0, 1, 2, 3, 4,5, 6, 7, or 8 amino acid substitutions, such as conservative amino acidsubstitutions.

In certain embodiments, an anti-GPC3 Adnectin comprises the sequence setforth in SEQ ID NO: 3, wherein BC, DE and FG loops as represented by(X)_(v), (X)_(x), and (X)_(z), respectively, comprise BC, DE, and FGloops having the amino acid sequences of SEQ ID NOs: 58, 59 and 60,respectively.

In certain embodiments, an anti-GPC3 Adnectin comprises the sequence setforth in SEQ ID NO: 3, wherein BC, DE and FG loops as represented by(X)_(v), (X)_(x), and (X)_(z), respectively, have amino acid sequencesat least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the BC,DE or FG loop sequences set forth in SEQ ID NOs: 71, 72 and 73,respectively.

In some embodiments, the anti-GPC3 Adnectin comprises the amino acidsequence set forth in SEQ ID NO: 3, wherein the BC, DE, and FG loopscomprise the amino acid sequences set forth in SEQ ID NOs: 71, 72 and73, respectively, wherein the BC loop has 0, 1, 2, 3, 4, 5, or 6 aminoacid substitutions, such as conservative amino acid substitutions, andthe DE loop has 0, 1, 2 or 3 amino acid substitutions, such as aconservative amino acid substitution, and the FG loop has 0, 1, 2, 3, 4,5, 6, 7, or 8 amino acid substitutions, such as conservative amino acidsubstitutions.

In certain embodiments, an anti-GPC3 Adnectin comprises the sequence setforth in SEQ ID NO: 3, wherein BC, DE and FG loops as represented by(X)_(v), (X)_(x), and (X)_(z), respectively, comprise BC, DE, and FGloops having the amino acid sequences of SEQ ID NOs: 71, 72 and 73,respectively.

In certain embodiments, an anti-GPC3 Adnectin comprises the sequence setforth in SEQ ID NO: 3, wherein BC, DE and FG loops as represented by(X)_(x), (X)_(x), and (X)_(z), respectively, have amino acid sequencesat least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the BC,DE or FG loop sequences set forth in SEQ ID NOs: 84, 85 and 86,respectively.

In some embodiments, the anti-GPC3 Adnectin comprises the amino acidsequence set forth in SEQ ID NO: 3, wherein the BC, DE, and FG loopscomprise the amino acid sequences set forth in SEQ ID NOs: 84, 85 and86, respectively, wherein the BC loop has 0, 1, 2, 3, 4, 5, or 6 aminoacid substitutions, such as conservative amino acid substitutions, andthe DE loop has 0, 1, 2 or 3 amino acid substitutions, such as aconservative amino acid substitution, and the FG loop has 0, 1, 2, 3, 4,5, 6, 7, or 8 amino acid substitutions, such as conservative amino acidsubstitutions.

In certain embodiments, an anti-GPC3 Adnectin comprises the sequence setforth in SEQ ID NO: 3, wherein BC, DE and FG loops as represented by(X)_(v), (X)_(x), and (X)_(z), respectively, comprise BC, DE, and FGloops having the amino acid sequences of SEQ ID NOs: 84, 85 and 86,respectively.

In certain embodiments, an anti-GPC3 Adnectin comprises the sequence setforth in SEQ ID NO: 3, wherein BC, DE and FG loops as represented by(X)_(v), (X)_(x), and (X)_(z), respectively, have amino acid sequencesat least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the BC,DE or FG loop sequences set forth in SEQ ID NOs: 99, 100 and 101,respectively.

In some embodiments, the anti-GPC3 Adnectin comprises the amino acidsequence set forth in SEQ ID NO: 3, wherein the BC, DE, and FG loopscomprise the amino acid sequences set forth in SEQ ID NOs: 99, 100 and101, respectively, wherein the BC loop has 0, 1, 2, 3, 4, 5, or 6 aminoacid substitutions, such as conservative amino acid substitutions, andthe DE loop has 0, 1, 2 or 3 amino acid substitutions, such as aconservative amino acid substitution, and the FG loop has 0, 1, 2, 3, 4,5, 6, 7, or 8 amino acid substitutions, such as conservative amino acidsubstitutions.

In certain embodiments, an anti-GPC3 Adnectin comprises the sequence setforth in SEQ ID NO: 3, wherein BC, DE and FG loops as represented by(X)_(v), (X)_(x), and (X)_(z), respectively, comprise BC, DE, and FGloops having the amino acid sequences of SEQ ID NOs: 99, 100 and 101,respectively.

In some embodiments, the anti-GPC3 Adnectin comprises the amino acidsequence set forth in SEQ ID NO: 3, wherein the BC, DE, and FG loopscomprise the amino acid sequences set forth in SEQ ID NOs: 99, 100 and129, respectively, wherein the BC loop has 0, 1, 2, 3, 4, 5, or 6 aminoacid substitutions, such as conservative amino acid substitutions, andthe DE loop has 0, 1, 2 or 3 amino acid substitutions, such as aconservative amino acid substitution.

In certain embodiments, an anti-GPC3 Adnectin comprises the sequence setforth in SEQ ID NO: 3, wherein BC, DE and FG loops as represented by(X)_(v), (X)_(x), and (X)_(z), respectively, comprise BC, DE, and FGloops having the amino acid sequences of SEQ ID NOs: SEQ ID NOs: 99, 100and 129, respectively.

In some embodiments, the anti-GPC3 Adnectin comprises the amino acidsequence set forth in SEQ ID NO: 3, wherein the BC, DE, and FG loopscomprise the amino acid sequences set forth in SEQ ID NOs: 99, 100 and156, respectively, wherein the BC loop has 0, 1, 2, 3, 4, 5, or 6 aminoacid substitutions, such as conservative amino acid substitutions, andthe DE loop has 0, 1, 2 or 3 amino acid substitutions, such as aconservative amino acid substitution.

In certain embodiments, an anti-GPC3 Adnectin comprises the sequence setforth in SEQ ID NO: 3, wherein BC, DE and FG loops as represented by(X)_(v), (X)_(x), and (X)_(z), respectively, comprise BC, DE, and FGloops having the amino acid sequences of SEQ ID NOs: SEQ ID NOs: 99, 100and 156, respectively.

In some embodiments, the anti-GPC3 Adnectin comprises the amino acidsequence set forth in SEQ ID NO: 3, wherein the BC, DE, and FG loopscomprise the amino acid sequences set forth in SEQ ID NOs: 99, 100 and183, respectively, wherein the BC loop has 0, 1, 2, 3, 4, 5, or 6 aminoacid substitutions, such as conservative amino acid substitutions, andthe DE loop has 0, 1, 2 or 3 amino acid substitutions, such as aconservative amino acid substitution.

In certain embodiments, an anti-GPC3 Adnectin comprises the sequence setforth in SEQ ID NO: 3, wherein BC, DE and FG loops as represented by(X)_(v), (X)_(x), and (X)_(z), respectively, comprise BC, DE, and FGloops having the amino acid sequences of SEQ ID NOs: SEQ ID NOs: 99, 100and 183, respectively.

In some embodiments, the anti-GPC3 Adnectin comprises the amino acidsequence set forth in SEQ ID NO: 3, wherein the BC, DE, and FG loopscomprise the amino acid sequences set forth in SEQ ID NOs: 99, 100 and210, respectively, wherein the BC loop has 0, 1, 2, 3, 4, 5, or 6 aminoacid substitutions, such as conservative amino acid substitutions, andthe DE loop has 0, 1, 2 or 3 amino acid substitutions, such as aconservative amino acid substitution.

In certain embodiments, an anti-GPC3 Adnectin comprises the sequence setforth in SEQ ID NO: 3, wherein BC, DE and FG loops as represented by(X)_(v), (X)_(x), and (X)_(z), respectively, comprise BC, DE, and FGloops having the amino acid sequences of SEQ ID NOs: SEQ ID NOs: 99, 100and 210, respectively.

In some embodiments, the anti-GPC3 Adnectin comprises the amino acidsequence set forth in SEQ ID NO: 3, wherein the BC, DE, and FG loopscomprise the amino acid sequences set forth in SEQ ID NOs: 99, 100 and237, respectively, wherein the BC loop has 0, 1, 2, 3, 4, 5, or 6 aminoacid substitutions, such as conservative amino acid substitutions, andthe DE loop has 0, 1, 2 or 3 amino acid substitutions, such as aconservative amino acid substitution.

In certain embodiments, an anti-GPC3 Adnectin comprises the sequence setforth in SEQ ID NO: 3, wherein BC, DE and FG loops as represented by(X)_(v), (X)_(x), and (X)_(z), respectively, comprise BC, DE, and FGloops having the amino acid sequences of SEQ ID NOs: SEQ ID NOs: 99, 100and 237, respectively.

In some embodiments, the anti-GPC3 Adnectin comprises the amino acidsequence set forth in SEQ ID NO: 3, wherein the BC, DE, and FG loopscomprise the amino acid sequences set forth in SEQ ID NOs: 99, 100 and264, respectively, wherein the BC loop has 0, 1, 2, 3, 4, 5, or 6 aminoacid substitutions, such as conservative amino acid substitutions, andthe DE loop has 0, 1, 2 or 3 amino acid substitutions, such as aconservative amino acid substitution.

In certain embodiments, an anti-GPC3 Adnectin comprises the sequence setforth in SEQ ID NO: 3, wherein BC, DE and FG loops as represented by(X)_(v), (X)_(x), and (X)_(z), respectively, comprise BC, DE, and FGloops having the amino acid sequences of SEQ ID NOs: SEQ ID NOs: 99, 100and 264, respectively.

In some embodiments, the anti-GPC3 Adnectin comprises the amino acidsequence set forth in SEQ ID NO: 3, wherein the BC, DE, and FG loopscomprise the amino acid sequences set forth in SEQ ID NOs: 99, 100 and291, respectively, wherein the BC loop has 0, 1, 2, 3, 4, 5, or 6 aminoacid substitutions, such as conservative amino acid substitutions, andthe DE loop has 0, 1, 2 or 3 amino acid substitutions, such as aconservative amino acid substitution.

In certain embodiments, an anti-GPC3 Adnectin comprises the sequence setforth in SEQ ID NO: 3, wherein BC, DE and FG loops as represented by(X)_(v), (X)_(x), and (X)_(z), respectively, comprise BC, DE, and FGloops having the amino acid sequences of SEQ ID NOs: SEQ ID NOs: 99, 100and 291, respectively.

In some embodiments, the anti-GPC3 Adnectin comprises the amino acidsequence set forth in SEQ ID NO: 3, wherein the BC, DE, and FG loopscomprise the amino acid sequences set forth in SEQ ID NOs: 99, 100 and318, respectively, wherein the BC loop has 0, 1, 2, 3, 4, 5, or 6 aminoacid substitutions, such as conservative amino acid substitutions, andthe DE loop has 0, 1, 2 or 3 amino acid substitutions, such as aconservative amino acid substitution.

In certain embodiments, an anti-GPC3 Adnectin comprises the sequence setforth in SEQ ID NO: 3, wherein BC, DE and FG loops as represented by(X)_(v), (X)_(x), and (X)_(z), respectively, comprise BC, DE, and FGloops having the amino acid sequences of SEQ ID NOs: SEQ ID NOs: 99, 100and 318, respectively.

The scaffold regions of such anti-GPC3 Adnectins may comprise anywherefrom 0 to 20, from 0 to 15, from 0 to 10, from 0 to 8, from 0 to 6, from0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1substitutions, conservative substitutions, deletions or additionsrelative to the scaffold amino acids residues of SEQ ID NO: 3. Suchscaffold modifications may be made, so long as the anti-GPC3 Adnectin iscapable of binding GPC3 with a desired K_(D).

In certain embodiments, the anti-GPC3 Adnectin comprises an amino acidsequence at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%identical to that of an anti-GPC3 Adnectin disclosed herein and having,e.g., any one of SEQ ID NOs: 5, 18, 31, 44, 57, 70, 83 and 98.

In certain embodiments, the anti-GPC3 Adnectin comprises an amino acidsequence at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%identical to any one of SEQ ID NOs: 5, 9-18, 22-31, 35-44, 48-57, 61-70,74-83, 87-98, 102-128, 130-155, 157-182, 184-209, 211-236, 238-263,265-290, 292-317 and 319-343.

In certain embodiments, the anti-GPC3 Adnectins described hereincomprise an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99% or100% identical to the non-BC, DE, and FG loop regions of SEQ ID NOs: 3,5, 18, 31, 44, 57, 70, 83 or 98.

In certain embodiments, the anti-GPC3 Adnectin comprises BC, DE, and FGloops as set forth in SEQ ID NOs: 6, 7, and 8, respectively; and anamino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to the non-BC, DE, and FG loop regions of SEQ ID NOs: 3, 5,18, 31, 44, 57, 70, 83 or 98.

In certain embodiments, the anti-GPC3 Adnectin comprises BC, DE, and FGloops as set forth in SEQ ID NOs: 19, 20 and 21, respectively; and anamino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to the non-BC, DE, and FG loop regions of SEQ ID NOs: 3, 5,18, 31, 44, 57, 70, 83 or 98.

In certain embodiments, the anti-GPC3 Adnectin comprises BC, DE, and FGloops as set forth in SEQ ID NOs: 32, 33 and 34, respectively; and anamino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to the non-BC, DE, and FG loop regions of SEQ ID NOs: 3, 5,18, 31, 44, 57, 70, 83 or 98.

In certain embodiments, the anti-GPC3 Adnectin comprises BC, DE, and FGloops as set forth in SEQ ID NOs: 45, 46 and 47, respectively; and anamino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to the non-BC, DE, and FG loop regions of SEQ ID NOs: 3, 5,18, 31, 44, 57, 70, 83 or 98.

In certain embodiments, the anti-GPC3 Adnectin comprises BC, DE, and FGloops as set forth in SEQ ID NOs: 58, 59 and 60, respectively and anamino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to the non-BC, DE, and FG loop regions of SEQ ID NOs: 3, 5,18, 31, 44, 57, 70, 83 or 98.

In certain embodiments, the anti-GPC3 Adnectin comprises BC, DE, and FGloops as set forth in SEQ ID NOs: 71, 72 and 73, respectively and anamino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to the non-BC, DE, and FG loop regions of SEQ ID NOs: 3, 5,18, 31, 44, 57, 70, 83 or 98.

In certain embodiments, the anti-GPC3 Adnectin comprises BC, DE, and FGloops as set forth in SEQ ID NOs: 84, 85 and 86, respectively and anamino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to the non-BC, DE, and FG loop regions of SEQ ID NOs: 3, 5,18, 31, 44, 57, 70, 83 or 98.

In certain embodiments, the anti-GPC3 Adnectin comprises BC, DE, and FGloops as set forth in SEQ ID NOs: 99, 100 and 101, respectively and anamino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to the non-BC, DE, and FG loop regions of SEQ ID NOs: 3, 5,18, 31, 44, 57, 70, 83 or 98.

In certain embodiments, the anti-GPC3 Adnectin comprises BC, DE, and FGloops as set forth in SEQ ID NOs: 99, 100 and 129, respectively and anamino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to the non-BC, DE, and FG loop regions of SEQ ID NOs: 3, 5,18, 31, 44, 57, 70, 83 or 98.

In certain embodiments, the anti-GPC3 Adnectin comprises BC, DE, and FGloops as set forth in SEQ ID NOs: 99, 100 and 129, respectively and anamino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to the non-BC, DE, and FG loop regions of SEQ ID NOs: 3, 5,18, 31, 44, 57, 70, 83 or 98.

In certain embodiments, the anti-GPC3 Adnectin comprises BC, DE, and FGloops as set forth in SEQ ID NOs: 99, 100 and 156, respectively and anamino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to the non-BC, DE, and FG loop regions of SEQ ID NOs: 3, 5,18, 31, 44, 57, 70, 83 or 98.

In certain embodiments, the anti-GPC3 Adnectin comprises BC, DE, and FGloops as set forth in SEQ ID NOs: 99, 100 and 183, respectively and anamino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to the non-BC, DE, and FG loop regions of SEQ ID NOs: 3, 5,18, 31, 44, 57, 70, 83 or 98.

In certain embodiments, the anti-GPC3 Adnectin comprises BC, DE, and FGloops as set forth in SEQ ID NOs: 99, 100 and 210, respectively and anamino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to the non-BC, DE, and FG loop regions of SEQ ID NOs: 3, 5,18, 31, 44, 57, 70, 83 or 98.

In certain embodiments, the anti-GPC3 Adnectin comprises BC, DE, and FGloops as set forth in SEQ ID NOs: 99, 100 and 237, respectively and anamino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to the non-BC, DE, and FG loop regions of SEQ ID NOs: 3, 5,18, 31, 44, 57, 70, 83 or 98.

In certain embodiments, the anti-GPC3 Adnectin comprises BC, DE, and FGloops as set forth in SEQ ID NOs: 99, 100 and 264, respectively and anamino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to the non-BC, DE, and FG loop regions of SEQ ID NOs: 3, 5,18, 31, 44, 57, 70, 83 or 98.

In certain embodiments, the anti-GPC3 Adnectin comprises BC, DE, and FGloops as set forth in SEQ ID NOs: 99, 100 and 291, respectively and anamino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to the non-BC, DE, and FG loop regions of SEQ ID NOs: 3, 5,18, 31, 44, 57, 70, 83 or 98.

In certain embodiments, the anti-GPC3 Adnectin comprises BC, DE, and FGloops as set forth in SEQ ID NOs: 99, 100 and 318, respectively and anamino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to the non-BC, DE, and FG loop regions of SEQ ID NOs: 3, 5,18, 31, 44, 57, 70, 83 or 98.

In some embodiments, the anti-GPC3 Adnectin comprises an amino acidsequence selected from the group consisting of 5, 18, 31, 44, 57, 70,83, 98, 128, 155, 182, 209, 209, 236, 263, 290 and 317.

In some embodiments, anti-GPC3 Adnectin further comprises a C-terminalmoiety selected from the group consisting of P_(m)X_(n), P_(m)CX_(n) andP_(m)CX_(n1)CX_(n2), wherein X is any amino acid and m, n, n1 and n2 areindependently 0 or an integer of 1, 2, 3, 4, 5 or more.

In some embodiments, the anti-GPC3 Adnectin comprises an amino acidsequence selected from the group consisting of 5, 9-18, 22-31, 35-44,48-57, 61-70, 74-83, 87-98, 102-128, 130-155, 157-182, 184-209, 211-236,238-263, 265-290, 292-317 and 319-343.

In certain embodiments, anti-GPC3 Adnectin comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 98, 102-128,129-155, 157-182, 184-209, 211-236, 238-263, 265-290, 292-317 and319-343, optionally with one or more histidines (e.g., 6×His) at theC-terminus.

In certain embodiments, the anti-GPC3 Adnectin comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 102-127. Incertain embodiments, the anti-GPC3 Adnectin comprises SEQ ID NOs:114-118. In other embodiments, the anti-GPC3 Adnectin comprises SEQ IDNOs: 123-127.

Provided herein are polypeptides that bind specifically to human GPC3with a KD of 10⁻⁷ or less, wherein the polypeptides comprise a ¹⁰Fn3domain comprising the BC, DE and FG loops of ADX_6077_A01, i.e., SEQ IDNOs: 99, 100 and 101. Provided herein are polypeptides that bindspecifically to human GPC3 with a KD of 10⁻⁷ or less, wherein thepolypeptides comprise a 10Fn3 domain comprising a BC loop comprising SEQID NO: 99, a DE loop comprising SEQ ID NO: 100 and an FG loop comprisingSEQ ID NO: 101. Also provided are polypeptides that bind specifically tohuman GPC3 with a KD of 10⁻⁷ or less, wherein the polypeptides comprisea ¹⁰Fn3 domain comprising a BC loop comprising SEQ ID NO: 99, a DE loopcomprising SEQ ID NO: 100 and an FG loop comprising SEQ ID NO: 101,wherein one of the two amino acid residues DG in the FG loop issubstituted with another amino acid. Also provided are polypeptides thatbind specifically to human GPC3 with a KD of 10⁻⁷ or less, wherein thepolypeptides comprise a ¹⁰Fn3 domain comprising a BC loop comprising SEQID NO: 99, a DE loop comprising SEQ ID NO: 100 and an FG loop comprisingSEQ ID NO: 101, wherein amino acid residue D of DG in the FG loop (i.e.,D78; the numbering is relative to that in SEQ ID NO: 102) is substitutedwith another amino acid, e.g., E, S, A, and G. Also provided arepolypeptides that bind specifically to human GPC3 with a KD of 10⁻⁷ orless, wherein the polypeptides comprise a ¹⁰Fn3 domain comprising a BCloop comprising SEQ ID NO: 99, a DE loop comprising SEQ ID NO: 100 andan FG loop comprising SEQ ID NO: 101, wherein amino acid residue G of DGin the FG loop (i.e., D79) is substituted with another amino acidresidue, e.g., S, A, L or V. Also provided are polypeptides that bindspecifically to human GPC3 with a KD of 10⁻⁷ or less, wherein thepolypeptides comprise a ¹⁰Fn3 domain comprising a BC loop comprising SEQID NO: 99, a DE loop comprising SEQ ID NO: 100 and an FG loop comprisingSEQ ID NO: 101, 129, 156, 183, 210, 237, 264, 291 or 318. Any of thepolypeptides described in this paragraph may comprise a cysteine residuelinked directly or indirectly to the C-terminal end of the polypeptideand/or may comprise one of the following amino acid residues orsequences linked directly or indirectly to the C-terminal end of thepolypeptide: P, PC, PCHHHHHH (SEQ ID NO: 395), PCPPPPPC (SEQ ID NO: 416)or PCPPPPPCHHHHHH (SEQ ID NO: 424).

Also provided are polypeptides that bind specifically to human GPC3 witha KD of 10⁻⁷ or less, wherein the polypeptides comprise a ¹Fn3 domaincomprising the ADX_6077_A01 core sequence, i.e., SEQ ID NO: 98, or anamino acid sequence that is at least 90%, 95%, 97%, 98%, or 99%identical thereto or that differs therefrom in 1-10, 1-5, 1-3, 1-2 or 1amino acid substitution (e.g., conservative amino acid substitutions),deletions or additions. Also provided are polypeptides that bindspecifically to human GPC3 with a KD of 10⁻⁷ or less, wherein thepolypeptides comprise a ¹Fn3 domain comprising the ADX_6077_A01 coresequence, i.e., SEQ ID NO: 98, or an amino acid sequence that is atleast 90%, 95%, 97%, 98%, or 99% identical thereto or that differstherefrom in 1-10, 1-5, 1-3, 1-2 or 1 amino acid substitution (e.g.,conservative amino acid substitutions), deletions or additions, and the¹Fn3 domain comprises a BC loop comprising SEQ ID NO: 99, a DE loopcomprising SEQ ID NO: 100 and an FG loop comprising SEQ ID NO: 101, orwhich differs therefrom in one amino acid of DG. Also provided arepolypeptides that bind specifically to human GPC3 with a KD of 10⁻⁷ orless, wherein the polypeptides comprise a ¹Fn3 domain comprising SEQ IDNO: 98, or an amino acid sequence that is at least 90%, 95%, 97%, 98%,or 99% identical thereto or that differs therefrom in 1-10, 1-5, 1-3,1-2 or 1 amino acid substitution (e.g., conservative amino acidsubstitutions), deletions or additions, and the ¹Fn3 domain comprises aBC loop comprising SEQ ID NO: 99, a DE loop comprising SEQ ID NO: 100and an FG loop comprising SEQ ID NO: 101, 129, 156, 183, 210, 237, 264,291 or 318. Also provided are polypeptides that bind specifically tohuman GPC3 with a KD of 10⁻⁷ or less, wherein the polypeptides comprisea ¹Fn3 domain comprising the ADX_6077_A01 core sequence, i.e., SEQ IDNO: 98, or an amino acid sequence that is at least 90%, 95%, 97%, 98%,or 99% identical thereto or that differs therefrom in 1-10, 1-5, 1-3,1-2 or 1 amino acid substitution (e.g., conservative amino acidsubstitutions), deletions or additions, and further comprises a cysteineresidue linked directly or indirectly to the C-terminal end of thepolypeptide. Also provided are polypeptides that bind specifically tohuman GPC3 with a KD of 10⁻⁷ or less, wherein the polypeptides comprisea ¹Fn3 domain comprising the ADX_6077_A01 core sequence, i.e., SEQ IDNO: 98, or an amino acid sequence that is at least 90%, 95%, 97%, 98%,or 99% identical thereto or that differs therefrom in 1-10, 1-5, 1-3,1-2 or 1 amino acid substitution (e.g., conservative amino acidsubstitutions), deletions or additions, and further comprises one of thefollowing amino acid residues or sequences linked directly or indirectlyto the C-terminal end of the polypeptide: P, PC, PCHHHHHH (SEQ ID NO:395), PCPPPPPC (SEQ ID NO: 416) or PCPPPPPCHHHHHH (SEQ ID NO: 424).

Provided herein are polypeptides comprising the amino acid sequence ofADX_6077_A01 or ADX_6912_G02 (with or without an N-terminal methionine)and with or without a 6×His tail.

Also provided herein are ¹⁰Fn3 proteins that bind specifically to humanGPC3 with a KD of 10⁻⁷ or less, and comprise the BC, DE and FG loops ofADX_6077_A01, i.e., SEQ ID NOs: 99, 100 and 101. Provided herein are¹⁰Fn3 proteins that bind specifically to human GPC3 with a KD of 10⁻⁷ orless, and comprise a BC loop comprising SEQ ID NO: 99, a DE loopcomprising SEQ ID NO: 100 and an FG loop comprising SEQ ID NO: 101. Alsoprovided are ° Fn3 proteins that bind specifically to human GPC3 with aKD of 10⁻⁷ or less, and comprise a BC loop comprising SEQ ID NO: 99, aDE loop comprising SEQ ID NO: 100 and an FG loop comprising SEQ ID NO:101, wherein one of the two amino acid residues DG in the FG loop issubstituted with another amino acid. Also provided are ¹⁰Fn3 proteinsthat bind specifically to human GPC3 with a KD of 10⁻⁷ or less, andcomprise a BC loop comprising SEQ ID NO: 99, a DE loop comprising SEQ IDNO: 100 and an FG loop comprising SEQ ID NO: 101, wherein amino acidresidue D of DG in the FG loop (i.e., D78; the numbering is relative tothat in SEQ ID NO: 102) is substituted with another amino acid, e.g., E,S, A, and G. Also provided are ° Fn3 proteins that bind specifically tohuman GPC3 with a KD of 10⁻⁷ or less, and comprise a BC loop comprisingSEQ ID NO: 99, a DE loop comprising SEQ ID NO: 100 and an FG loopcomprising SEQ ID NO: 101, wherein amino acid residue G of DG in the FGloop (i.e., D79) is substituted with another amino acid residue, e.g.,S, A, L or V. Also provided are ¹⁰Fn3 proteins that bind specifically tohuman GPC3 with a KD of 10⁻⁷ or less, and comprise a BC loop comprisingSEQ ID NO: 99, a DE loop comprising SEQ ID NO: 100 and an FG loopcomprising SEQ ID NO: 101, 129, 156, 183, 210, 237, 264, 291 or 318. Anyof the ¹⁰Fn3 proteins described in this paragraph may comprise acysteine residue linked directly or indirectly to its C-terminus and/ormay comprise one of the following amino acid residues or sequenceslinked directly or indirectly to the C-terminus: P, PC, PCHHHHHH (SEQ IDNO: 395), PCPPPPPC (SEQ ID NO: 416) or PCPPPPPCHHHHHH (SEQ ID NO: 424).

Also provided are ¹⁰Fn3 proteins that bind specifically to human GPC3with a KD of 10⁷′ or less, and comprise the ADX_6077_A01 core sequence,i.e., SEQ ID NO: 98, or an amino acid sequence that is at least 90%,95%, 97%, 98%, or 99% identical thereto or that differs therefrom in1-10, 1-5, 1-3, 1-2 or 1 amino acid substitution (e.g., conservativeamino acid substitutions), deletions or additions. Also provided are¹Fn3 proteins that bind specifically to human GPC3 with a KD of 10⁻⁷ orless, and comprise the ADX_6077_A01 core sequence, i.e., SEQ ID NO: 98,or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99%identical thereto or that differs therefrom in 1-10, 1-5, 1-3, 1-2 or 1amino acid substitution (e.g., conservative amino acid substitutions),deletions or additions, and comprise a BC loop comprising SEQ ID NO: 99,a DE loop comprising SEQ ID NO: 100 and an FG loop comprising SEQ ID NO:101, or which differs therefrom in one amino acid of DG. Also providedare ¹⁰Fn3 proteins that bind specifically to human GPC3 with a KD of10⁻⁷ or less, and comprise SEQ ID NO: 98, or an amino acid sequence thatis at least 90%, 95%, 97%, 98%, or 99% identical thereto or that differstherefrom in 1-10, 1-5, 1-3, 1-2 or 1 amino acid substitution (e.g.,conservative amino acid substitutions), deletions or additions, andcomprise a BC loop comprising SEQ ID NO: 99, a DE loop comprising SEQ IDNO: 100 and an FG loop comprising SEQ ID NO: 101, 129, 156, 183, 210,237, 264, 291 or 318. Also provided are ¹⁰Fn3 proteins that bindspecifically to human GPC3 with a KD of 10⁻⁷ or less, and comprise a¹⁰Fn3 domain comprising the ADX_6077_A01 core sequence, i.e., SEQ ID NO:98, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or99% identical thereto or that differs therefrom in 1-10, 1-5, 1-3, 1-2or 1 amino acid substitution (e.g., conservative amino acidsubstitutions), deletions or additions, and further comprises a cysteineresidue linked directly or indirectly to its C-terminus. Also providedare ¹⁰Fn3 proteins that bind specifically to human GPC3 with a KD of10⁷′ or less, and comprise a ¹⁰Fn3 domain comprising the ADX_6077_A01core sequence, i.e., SEQ ID NO: 98, or an amino acid sequence that is atleast 90%, 95%, 97%, 98%, or 99% identical thereto or that differstherefrom in 1-10, 1-5, 1-3, 1-2 or 1 amino acid substitution (e.g.,conservative amino acid substitutions), deletions or additions, andfurther comprises one of the following amino acid residues or sequenceslinked directly or indirectly to its C-terminus: P, PC, PCHHHHHH (SEQ IDNO: 395), PCPPPPPC (SEQ ID NO: 416) or PCPPPPPCHHHHHH (SEQ ID NO: 424).

Provided herein are ¹⁰Fn3 proteins comprising the amino acid sequence ofADX_6077_A01 or ADX_6912_G02 (with or without an N-terminal methionine)and with or without a 6×His tail.

Also provided are drug conjugates comprising one of the polypeptides or¹⁰Fn3 proteins described in the above paragraphs, conjugated to a drugmoiety, such as a tubulysin analog.

Further provided are ¹⁰Fn3 proteins or polypeptides comprising ¹⁰Fn3domains comprising at their C-terminus a sequence comprising one or morecysteines, wherein at least one cysteine is conjugated to a tubulysinanalog described herein. For example, ¹⁰Fn3 proteins or polypeptidescomprising ¹⁰Fn3 domains may be linked to a peptide comprising the aminoacid sequence PmCn, wherein m and n are independent an integer of 1 ormore and wherein one or more cysteines is conjugated to a tubulysinanalog described herein.

In certain embodiments, BC, DE and/or FG loop amino acid sequences ofany one of the anti-GPC3 Adnectins (e.g., SEQ ID NOs: 5, 9-18, 22-31,35-44, 48-57, 61-70, 74-83, 87-98, 102-128, 130-155, 157-182, 184-209,211-236, 238-263, 265-290, 292-317 and 319-343) described herein aregrafted into non-¹⁰Fn3 domain protein scaffolds. For instance, one ormore loop amino acid sequences is exchanged for or inserted into one ormore CDR loops of an antibody heavy or light chain or fragment thereof.In other embodiments, the protein domain into which one or more aminoacid loop sequences are exchanged or inserted includes, but is notlimited to, consensus Fn3 domains (Centocor, US), ankyrin repeatproteins (Molecular Partners AG, Zurich Switzerland), domain antibodies(Domantis, Ltd, Cambridge, Mass.), single domain camelid nanobodies(Ablynx, Belgium), lipocalins (e.g., anticalins; Pieris Proteolab AG,Freising, Germany), Avimers (Amgen, CA), affibodies (Affibody AG,Sweden), ubiquitin (e.g., affilins; Scil Proteins GmbH, Halle, Germany),protein epitope mimetics (Polyphor Ltd, Allschwil, Switzerland), helicalbundle scaffolds (e.g. alphabodies, Complix, Belgium), Fyn SH3 domains(Covagen AG, Switzerland), or atrimers (Anaphor, Inc., CA).

B. Cross-Competing Anti-GPC3 Adnectins

Also provided are Adnectins that compete (e.g., cross-compete) forbinding to human GPC3 with the particular anti-GPC3 Adnectins describedherein. Such competing Adnectins can be identified based on theirability to competitively inhibit binding to GPC3 of Adnectins describedherein in standard GPC3 binding assays. For example, standard ELISAassays can be used in which a recombinant GPC3 protein is immobilized onthe plate, one of the Adnectins is fluorescently labeled and the abilityof non-labeled Adnectins to compete off the binding of the labeledAdnectin is evaluated.

In certain embodiments, a competitive ELISA format can be performed todetermine whether two anti-GPC3 Adnectins bind overlapping Adnectinbinding sites on GPC3. In one format, Adnectin #1 is coated on a plate,which is then blocked and washed. To this plate is added either GPC3alone, or GPC3 pre-incubated with a saturating concentration of Adnectin#2. After a suitable incubation period, the plate is washed and probedwith a polyclonal anti-GPC3 antibody, such as a biotinylated anti-GPC3polyclonal antibody, followed by detection with streptavidin-HRPconjugate and standard tetramethylbenzidine development procedures. Ifthe OD signal is the same with or without preincubation with Adnectin#2, then the two Adnectins bind independently of one another, and theirAdnectin binding sites do not overlap. If, however, the OD signal forwells that received GPC3/Adnectin #2 mixtures is lower than for thosethat received GPC3 alone, then binding of Adnectin #2 is confirmed toblock binding of Adnectin #1 to GPC3.

Alternatively, a similar experiment is conducted by surface plasmonresonance (SPR, e.g., BIAcore). Adnectin #1 is immobilized on an SPRchip surface, followed by injections of either GPC3 alone or GPC3pre-incubated with a saturating concentration of Adnectin #2. If thebinding signal for GPC3/Adnectin #2 mixtures is the same or higher thanthat of GPC3 alone, then the two Adnectins bind independently of oneanother, and their Adnectin binding sites do not overlap. If, however,the binding signal for GPC3/Adnectin #2 mixtures is lower than thebinding signal for GPC3 alone, then binding of Adnectin #2 is confirmedto block binding of Adnectin #1 to GPC3. A feature of these experimentsis the use of saturating concentrations of Adnectin #2. If GPC3 is notsaturated with Adnectin #2, then the conclusions above do not hold.Similar experiments can be used to determine if any two GPC3 bindingproteins bind to overlapping Adnectin binding sites.

Both assays exemplified above may also be performed in the reverse orderwhere Adnectin #2 is immobilized and GPC3-Adnectin #1 are added to theplate. Alternatively, Adnectin #1 and/or #2 can be replaced with amonoclonal antibody and/or soluble receptor-Fc fusion protein.

In another embodiment, competition can be determined using a HTRFsandwich assay.

Candidate competing anti-GPC3 Adnectins can inhibit the binding of ananti-GPC3 Adnectin described herein to GPC3 by at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 97%, at least98%, or at least 99% and/or their binding is inhibited by at least 50%,at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 97%, atleast 98%, or at least 99%. The % competition can be determined usingthe methods described above.

In some embodiments, molecules that compete with the anti-GPC3 Adnectinsdescribed herein need not be an Adnectin, but can be any type ofmolecule that binds to GPC3, such as, but not limited to, an antibody, asmall molecule, a peptide, and the like.

In certain embodiments, an Adnectin binds to the same Adnectin bindingsite on GPC3 as a particular anti-GPC3 Adnectin described herein.Standard mapping techniques, such as protease mapping, mutationalanalysis, HDX-MS, x-ray crystallography and 2-dimensional nuclearmagnetic resonance, can be used to determine whether an Adnectin bindsto the same Adnectin binding site as a reference Adnectin (see, e.g.,Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996)).

In some embodiments, anti-GPC3 Adnectins provided herein bind to adiscontinuous Adnectin binding site on human GPC3. In some embodiments,an anti-GPC3 FBS binds a region of, e.g., 10-20 amino acid residues,within human GPC3 (SEQ ID NO:344) which comprises SEQ ID NO: 345. Insome embodiments, an anti-GPC3 Adnectin binds a region of, e.g., 10-20amino acid residues, within human GPC3 (SEQ ID NO: 344) which comprisesSEQ ID NO: 346. In other embodiments, an anti-GPC3 FBS binds two regionsof, e.g., 10-20 amino acid residues, each within human GPC3 (SEQ ID NO:344), one comprising SEQ ID NO: 345 and the other region comprising SEQID NO: 346, respectively.

C. N-Terminal and C-Terminal Modified Anti-GPC3 Adnectins

In some embodiments, the amino acid sequences of the N-terminal and/orC-terminal regions of an Adnectin are modified by deletion, substitutionor insertion relative to the amino acid sequences of the correspondingregions of ¹⁰Fn3 domains comprising, e.g., SEQ ID NO: 1.

In certain embodiments, the amino acid sequence of the first 1, 2, 3, 4,5, 6, 7, 8 or 9 residues of Adnectins, e.g., having sequences startingwith “VSD”, as in, e.g., SEQ ID NO: 1, may be modified or deleted in thepolypeptides provided herein. In exemplary embodiments, the amino acidscorresponding to amino acids 1-7, 8 or 9 of Adnectins having sequencesstarting with “VSD”, as in, e.g., SEQ ID NO: 1 are replaced with analternative N-terminal region having from 1-20, 1-15, 1-10, 1-8, 1-5,1-4, 1-3, 1-2, or 1 amino acids in length.

Exemplary alternative N-terminal regions that can be added to GPC3Adnectin core sequences or those starting with “VSD” include(represented by the single letter amino acid code) M, MG, G, MGVSDVPRD(SEQ ID NO: 351) and GVSDVPRD (SEQ ID NO: 352). Other suitablealternative N-terminal regions include, for example, X_(n)SDVPRDL (SEQID NO: 353), X_(n)DVPRDL (SEQ ID NO: 354), X_(n)VPRDL (SEQ ID NO: 355),X_(n)PRDL (SEQ ID NO: 356), X_(n)RDL (SEQ ID NO: 357), X_(n)DL (SEQ IDNO: 358), or X_(n)L, wherein n=0, 1 or 2 amino acids, wherein when n=1,X is Met or Gly, and when n=2, X is Met-Gly. When a Met-Gly sequence isadded to the N-terminus of a ¹⁰Fn3 domain, the M will usually be cleavedoff, leaving a G at the N-terminus. In other embodiments, thealternative N-terminal region comprises the amino acid sequence MASTSG(SEQ ID NO: 359). In certain embodiments, the N-terminal extensionconsists of an amino acid sequence selected from the group consistingof: M, MG, and G.

In some embodiments, an alternative C-terminal region having from 1-20,1-15, 1-10, 1-8, 1-5, 1-4, 1-3, 1-2, or 1 amino acids in length can beadded to the C-terminal region of GPC3 Adnectins ending in “RT”, as,e.g., in SEQ ID NO: 1. Examples of alternative C-terminal regionsequences include, for example, polypeptides comprising, consistingessentially of, or consisting of, EIEK (SEQ ID NO: 360), EGSGC (SEQ IDNO: 361), EIEKPCQ (SEQ ID NO: 362), EIEKPSQ (SEQ ID NO: 363), EIEKP (SEQID NO: 364), EIEKPS (SEQ ID NO: 365), EIEKPC (SEQ ID NO: 366), EIDK (SEQID NO: 367), EIDKPCQ (SEQ ID NO: 368) or EIDKPSQ (SEQ ID NO: 369). Incertain embodiments, the C-terminal region consists of EIDKPCQ (SEQ IDNO: 368). In certain embodiments, ¹⁰Fn3 domain is linked to a C-terminalextension sequence that comprises E and D residues, and may be between 8and 50, 10 and 30, 10 and 20, 5 and 10, and 2 and 4 amino acids inlength. In some embodiments, tail sequences include ED-based linkers inwhich the sequence comprises tandem repeats of ED. In exemplaryembodiments, the tail sequence comprises 2-10, 2-7, 2-5, 3-10, 3-7, 3-5,3, 4 or 5 ED repeats. In certain embodiments, the ED-based tailsequences may also include additional amino acid residues, such as, forexample: EI, EID, ES, EC, EGS, and EGC. Such sequences are based, inpart, on known Adnectin tail sequences, such as EIDKPSQ (SEQ ID NO:369), in which residues D and K have been removed. In some embodiments,the ED-based tail comprises an E, I or E1 residues before the EDrepeats.

In certain embodiments, the N- or C-terminal extension sequences arelinked to the ¹⁰Fn3 domain with known linker sequences (e.g., SEQ IDNOs:426-451 in Table 13). In some embodiments, sequences may be placedat the C-terminus of the ¹⁰Fn3 domain to facilitate attachment of apharmacokinetic moiety. For example, a cysteine containing linker suchas GSGC may be added to the C-terminus to facilitate site directedPEGylation on the cysteine residue.

In certain embodiments, an alternative C-terminal moiety, which can belinked to the C-terminal amino acids RT (i.e., amino acid 94, e.g., asin SEQ ID NO: 1) of GPC3 Adnectins comprises the amino acids P_(m)X_(n),wherein P is proline, X is any amino acid, m is an integer that is atleast 1 and n is 0 or an integer that is at least 1. In someembodiments, m may be 1, 2, 3 or more. For example, m may be 1-3 or mmay be 1-2. “n” may be 0, 1, 2, 3 or more, e.g., n may be 1-3 or 1-2.

The P_(m)X_(n) moiety may be linked directly to the C-terminal aminoacid of a ¹⁰Fn3 moiety, e.g., to its 94^(th) amino acid (based on aminoacid numbering of SEQ ID NO: 1). The P_(m)X_(n) moiety may be linked viaa peptide bond to the 94^(th) amino acid of a ¹⁰Fn3 moiety. A singleproline residue at the end of SEQ ID NO: 1 is referred to as “95Pro” or“Pro95” or “P95” or “95P”.

In certain embodiments, n is not 0, and may be, e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, 10 or more. For example, n may be from 0-10, 0-5, 0-3, 1-10,1-5, 1-3 or 1-2. However, more than 10 amino acids may be linked to theproline. For example, in a tandem FBS moiety or a FBS moiety fused toanother polypeptide, the C-terminal amino acid of the FBS moiety may belinked to one or more prolines, and the last proline is linked to thesecond FBS moiety or to the heterologous moiety. Therefore, in certainembodiments, n may be an integer ranging from 0-100, 0-200, 0-300,0-400, 0-500 or more.

In certain embodiments, P_(m)X_(n) linked to the C-terminus of a GPC3Adnectin comprises a cysteine. For example, the first amino acid afterthe proline may be a cysteine, and the cysteine may be the last aminoacid in the molecule or the cysteine may be followed by one or moreamino acids. The presence of a cysteine permits the conjugation ofheterologous moieties to the FBS moiety, e.g., chemical moieties, e.g.,PEG. Exemplary P_(m)X_(n) moieties comprising a cysteine include:P_(m)CX_(n), wherein C is a cysteine, each X is independently any aminoacid, m is an integer that is at least 1 and n is 0 or an integer thatis at least 1. In some embodiments, m may be 1, 2, 3 or more. Forexample, m may be 1-3 or m may be 1-2. “n” may be 0, 1, 2, 3 or more,e.g., n may be 1-3 or 1-2. Other exemplary P_(m)X_(n) moieties includetwo cysteines, for example, P_(m)CX_(n1)CX_(n2), wherein each X isindependently any amino acid, n₁ and n₂ are independently 0 or aninteger that is at least 1. For example, n₁ may be 1, 2, 3, 4 or 5 andn₂ may be 1, 2, 3, 4 or 5. Exemplary P_(m)X_(n) moieties include thoselisted in Table 1.

TABLE 1 Exemplary PmXn moieties Moieties with 1 prolineMoieties with 2 or more prolines Moieties with 2 cysteines P PP PCC PIPPI PCGC (SEQ ID NO: 412) PC PPC PCPC (SEQ ID NO: 413) PIDPPID (SEQ ID NO: 396) PCGSGC (SEQ ID NO: 414) PIE PPIE (SEQ ID NO: 397)PCPPPC (SEQ ID NO: 415) PIDK (SEQ ID NO: 382) PPIDK (SEQ ID NO: 398)PCPPPPPC (SEQ ID NO: 416) PIEK (SEQ ID NO: 383) PPIEK (SEQ ID NO: 399)PCGSGSGC (SEQ ID NO: 417) PIDKP (SEQ ID NO: 384) PPIDKP (SEQ ID NO: 400)PCHHHHHC (SEQ ID NO: 418) PIEKP (SEQ ID NO: 385) PPIEKP (SEQ ID NO: 401)PCCHHHHHH (SEQ ID NO: 419) PIDKPS (SEQ ID NO: 386)PPIDKPS (SEQ ID NO: 402) PCGCHHHHHH (SEQ ID NO: 420)PIEKPS (SEQ ID NO: 387) PPIEKPS (SEQ ID NO: 403)PCPCHHHHHH (SEQ ID NO: 421) PIDKPC (SEQ ID NO: 388)PPIDKPC (SEQ ID NO: 404) PCGSGCHHHHHH (SEQ ID NO: 422)PIEKPC (SEQ ID NO: 389) PPIEKPC (SEQ ID NO: 405)PCPPPCHHHHHH (SEQ ID NO: 423) PIDKPSQ (SEQ ID NO: 390)PPIDKPSQ (SEQ ID NO: 406) PCPPPPPCHHHHHH (SEQ ID NO: 424)PIEKPSQ (SEQ ID NO: 391) PPIEKPSQ (SEQ ID NO: 407)PCGSGSGCHHHHHH (SEQ ID NO: 425) PIDKPCQ (SEQ ID NO: 392)PPIDKPCQ (SEQ ID NO: 408) PIEKPCQ (SEQ ID NO: 393)PPIEKPCQ (SEQ ID NO: 409) PHHHHHH (SEQ ID NO: 394)PPHHHHHH (SEQ ID NO: 410) PCHHHHHH (SEQ ID NO: 395)PPCHHHHHH (SEQ ID NO: 411)

In certain embodiments, for example, the P_(m)X_(n) moiety is selectedfrom the group consisting of PC, PPC and PCC. In another embodiment, theP_(m)X_(n) moiety is P_(m)CXn₁CXn₂. In certain embodiments,P_(m)CXn₁CXn₂ is selected from the group consisting of PCPPPC (SEQ IDNO: 415) and PCPPPPPC (SEQ ID NO: 416).

Any of the C-terminal modifications described herein may be applied toGPC3 Adnectins.

Any of the P_(m)X_(n) moieties, e.g., those shown in Table 1 may befollowed by a histidine tail, e.g., 6×His tag, or other tag. This doesnot exclude that a histidine tail may be included in P_(m)X_(n).

In certain embodiments, the fibronectin based scaffold proteins comprisea ¹⁰Fn3 domain having both an alternative N-terminal region sequence andan alternative C-terminal region sequence, and optionally a 6×his tail.

II. Multivalent Polypeptides

In certain embodiments, a protein comprises GPC3 FBS and at least oneother FBS. A multivalent FBS may comprise 2, 3 or more FBS, that arecovalently associated. In exemplary embodiments, the FBS moiety is abispecific or dimeric protein comprising two ¹⁰Fn3 domains.

The FBS moieties, e.g., ¹⁰Fn3 domains, in a multivalent protein may beconnected by a polypeptide linker. Exemplary polypeptide linkers includepolypeptides having from 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, or 1-2amino acids. Suitable linkers for joining the ¹⁰Fn3 domains are thosewhich allow the separate domains to fold independently of each otherforming a three dimensional structure that permits high affinity bindingto a target molecule. Specific examples of suitable linkers includeglycine-serine based linkers, glycine-proline based linkers,proline-alanine based linkers as well as any other linkers describedherein. In some embodiments, the linker is a glycine-proline basedlinker. These linkers comprise glycine and proline residues and may bebetween 3 and 30, 10 and 30, and 3 and 20 amino acids in length.Examples of such linkers include GPG, GPGPGPG (SEQ ID NO: 436) andGPGPGPGPGPG (SEQ ID NO: 437). In some embodiments, the linker is aproline-alanine based linker. These linkers comprise proline and alanineresidues and may be between 3 and 30, 10 and 30, 3 and 20 and 6 and 18amino acids in length. Examples of such linkers include PAPAPA (SEQ IDNO: 438), PAPAPAPAPAPA (SEQ ID NO: 439) and PAPAPAPAPAPAPAPAPA (SEQ IDNO: 440). In some embodiments, the linker is a glycine-serine basedlinker. These linkers comprise glycine and serine residues and may bebetween 8 and 50, 10 and 30, and 10 and 20 amino acids in length.Examples of such linkers may contain, for example, (GS)₅₋₁₀ (SEQ ID NO:464), (G₄S)₂₋₅ (SEQ ID NO: 465), and (G₄S)₂G (SEQ ID NO: 466). Examplesof such linkers include SEQ ID NOs: 427-439. In exemplary embodiments,the linker does not contain any Asp-Lys (DK) pairs.

III. Pharmacokinteic Moieties

For therapeutic purposes, the anti-GPC3 Adnectins described herein maybe linked directly or indirectly to a pharmacokinetic (PK) moiety.Improved pharmacokinetics may be assessed according to the perceivedtherapeutic need. Often it is desirable to increase bioavailabilityand/or increase the time between doses, possibly by increasing the timethat a protein remains available in the serum after dosing. In someinstances, it is desirable to improve the continuity of the serumconcentration of the protein over time (e.g., decrease the difference inserum concentration of the protein shortly after administration andshortly before the next administration). The anti-GPC3 Adnectin may beattached to a moiety that reduces the clearance rate of the polypeptidein a mammal (e.g., mouse, rat, or human) by greater than two-fold,greater than three-fold, greater than four-fold or greater thanfive-fold relative to the unmodified anti-GPC3 Adnectin. Other measuresof improved pharmacokinetics may include serum half-life, which is oftendivided into an alpha phase and a beta phase. Either or both phases maybe improved significantly by addition of an appropriate moiety. Forexample, the PK moiety may increase the serum half-life of thepolypeptide by more than 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,120, 150, 200, 400, 600, 800, 1000% or more relative to the Fn3 domainalone.

Moieties that slow clearance of a protein from the blood, hereinreferred to as “PK moieties”, include polyoxyalkylene moieties (e.g.,polyethylene glycol), sugars (e.g., sialic acid), and well-toleratedprotein moieties (e.g., Fc and fragments and variants thereof,transferrin, or serum albumin). The anti-GPC3 Adnectin may also be fusedto albumin or a fragment (portion) or variant of albumin as described inU.S. Publication No. 2007/0048282, or may be fused to one or more serumalbumin binding Adnectin, as described herein.

Other PK moieties that can be used in the invention include thosedescribed in Kontermann et al., (Current Opinion in Biotechnology 2011;22:868-76), herein incorporated by reference. Such PK moieties include,but are not limited to, human serum albumin fusions, human serum albuminconjugates, human serum albumin binders (e.g., Adnectin PKE, AlbudAb,ABD), XTEN fusions, PAS fusions (i.e., recombinant PEG mimetics based onthe three amino acids proline, alanine, and serine), carbohydrateconjugates (e.g., hydroxyethyl starch (HES)), glycosylation, polysialicacid conjugates, and fatty acid conjugates.

In some embodiments the invention provides an anti-GPC3 Adnectin fusedto a PK moiety that is a polymeric sugar. In some embodiments, the PKmoiety is a polyethylene glycol moiety or an Fc region. In someembodiments the PK moiety is a serum albumin binding protein such asthose described in U.S. Publication Nos. 2007/0178082 and 2007/0269422.In some embodiments the PK moiety is human serum albumin. In someembodiments, the PK moiety is transferrin.

In some embodiments, the PK moiety is linked to the anti-GPC3 Adnectinvia a polypeptide linker. Exemplary polypeptide linkers includepolypeptides having from 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, or 1-2amino acids. Suitable linkers for joining the Fn3 domains are thosewhich allow the separate domains to fold independently of each otherforming a three dimensional structure that permits high affinity bindingto a target molecule. In exemplary embodiments, the linker does notcontain any Asp-Lys (DK) pairs. A list of suitable linkers is providedin Table 14 (e.g., SEQ ID NOs: 426-451).

In some embodiments, an anti-GPC3 Adnectin is linked, for example, to ananti-HSA Adnectin via a polypeptide linker having a protease site thatis cleavable by a protease in the blood or target tissue. Suchembodiments can be used to release an anti-GPC3 Adnectin for betterdelivery or therapeutic properties or more efficient production.

Additional linkers or spacers, may be introduced at the N-terminus orC-terminus of a Fn3 domain between the Fn3 domain and the polypeptidelinker.

Polyethylene Glycol

In some embodiments, the anti-GPC3 Adnectin comprises polyethyleneglycol (PEG). PEG is a well-known, water soluble polymer that iscommercially available or can be prepared by ring-opening polymerizationof ethylene glycol according to methods well known in the art (Sandlerand Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, pages138-161). The term “PEG” is used broadly to encompass any polyethyleneglycol molecule, without regard to size or to modification at an end ofthe PEG, and can be represented by the formula:X—O(CH₂CH₂O)_(n-1)CH₂CH₂OH, where n is 20 to 2300 and X is H or aterminal modification, e.g., a C₁₋₄ alkyl. PEG can contain furtherchemical groups which are necessary for binding reactions, which resultfrom the chemical synthesis of the molecule; or which act as a spacerfor optimal distance of parts of the molecule. In addition, such a PEGcan consist of one or more PEG side-chains which are linked together.PEGs with more than one PEG chain are called multiarmed or branchedPEGs. Branched PEGs are described in, for example, European PublishedApplication No. 473084A and U.S. Pat. No. 5,932,462.

Immunoglobulin Fc Domain (and Fragments)

In certain embodiments, the anti-GPC3 Adnectin is fused to animmunoglobulin Fc domain, or a fragment or variant thereof. As usedherein, a “functional Fc region” is an Fc domain or fragment thereofwhich retains the ability to bind FcRn. In some embodiments, afunctional Fc region binds to FcRn, bud does not possess effectorfunction. The ability of the Fc region or fragment thereof to bind toFcRn can be determined by standard binding assays known in the art. Inother embodiments, the Fc region or fragment thereof binds to FcRn andpossesses at least one “effector function” of a native Fc region.Exemplary “effector functions” include C1q binding; complement dependentcytotoxicity (CDC); Fc receptor binding; antibody-dependentcell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cellsurface receptors (e.g., B cell receptor; BCR), etc. Such effectorfunctions generally require the Fc region to be combined with a bindingdomain (e.g., an anti-GPC3 Adnectin) and can be assessed using variousassays known in the art for evaluating such antibody effector functions.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. A “variantFc region” comprises an amino acid sequence which differs from that of anative sequence Fc region by virtue of at least one amino acidmodification. Preferably, the variant Fc region has at least one aminoacid substitution compared to a native sequence Fc region or to the Fcregion of a parent polypeptide, e.g., from about one to about ten aminoacid substitutions, and preferably from about one to about five aminoacid substitutions in a native sequence Fc region or in the Fc region ofthe parent polypeptide. The variant Fc region herein will preferablypossess at least about 80% sequence identity with a native sequence Fcregion and/or with an Fc region of a parent polypeptide, and mostpreferably at least about 90% sequence identity therewith, morepreferably at least about 95% sequence identity therewith.

In an exemplary embodiment, the Fc domain is derived from an IgG1subclass, however, other subclasses (e.g., IgG2, IgG3, and IgG4) mayalso be used. Shown below is the sequence of a human IgG1 immunoglobulinFc domain:

(SEQ ID NO: 463) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The core hinge sequence is underlined, and the CH2 and CH3 regions arein regular text. It should be understood that the C-terminal lysine isoptional. Allotypes and mutants of this sequence may also be used. As isknown in the art, mutants can be designed to modulate a variety ofproperties of the Fc, e.g., ADCC, CDC or half-life.

In certain embodiments, the Fc region used in the anti-GPC3 Adnectinfusion comprises a 7 region. In certain embodiments, the Fc region usedin the anti-GPC3 Adnectin fusion comprises CH2 and CH3 regions. Incertain embodiments, the Fc region used in the anti-GPC3 Adnectin fusioncomprises a CH2, CH3, and hinge region.

In certain embodiments, the “hinge” region comprises the core hingeresidues spanning positions 1-16 of SEQ ID NO: 463 (DKTHTCPPCPAPELLG;SEQ ID NO: 464) of the IgG1 Fc region. In certain embodiments, theanti-GPC3 Adnectin-Fc fusion adopts a multimeric structure (e.g., dimer)owing, in part, to the cysteine residues at positions 6 and 9 of SEQ IDNO: within the hinge region.

IV. Vectors and Polynucleotides

Also provided herein are nucleic acids encoding the anti-GPC3 Andectinsdescribed herein. As appreciated by those skilled in the art, because ofthird base degeneracy, almost every amino acid can be represented bymore than one triplet codon in a coding nucleotide sequence. Inaddition, minor base pair changes may result in a conservativesubstitution in the amino acid sequence encoded but are not expected tosubstantially alter the biological activity of the gene product.Therefore, a nucleic acid sequence encoding a protein described hereinmay be modified slightly in sequence and yet still encode its respectivegene product. Certain exemplary nucleic acids encoding the anti-GPC3Adnectins and their fusions described herein include nucleic acidshaving the sequences set forth in SEQ ID NOs: 452-462.

Nucleic acids encoding any of the various proteins comprising ananti-GPC3 Adnectin disclosed herein may be synthesized chemically,enzymatically or recombinantly. Codon usage may be selected so as toimprove expression in a cell. Such codon usage will depend on the celltype selected. Specialized codon usage patterns have been developed forE. coli and other bacteria, as well as mammalian cells, plant cells,yeast cells and insect cells. See for example: Mayfield et al., Proc.Natl. Acad. Sci. USA, 100(2):438-442 (Jan. 21, 2003); Sinclair et al.,Protein Expr. Purif, 26(1):96-105 (October 2002); Connell, N. D., Curr.Opin. Biotechnol., 12(5):446-449 (October 2001); Makrides et al.,Microbiol. Rev., 60(3):512-538 (September 1996); and Sharp et al.,Yeast, 7(7):657-678 (October 1991).

General techniques for nucleic acid manipulation are described forexample in Sambrook et al., Molecular Cloning: A Laboratory Manual,Second Edition, Vols. 1-3, Cold Spring Harbor Laboratory Press (1989),or Ausubel, F. et al., Current Protocols in Molecular Biology, GreenPublishing and Wiley-Interscience, New York (1987) and periodic updates,herein incorporated by reference. The DNA encoding the polypeptide isoperably linked to suitable transcriptional or translational regulatoryelements derived from mammalian, viral, or insect genes. Such regulatoryelements include a transcriptional promoter, an optional operatorsequence to control transcription, a sequence encoding suitable mRNAribosomal binding sites, and sequences that control the termination oftranscription and translation. The ability to replicate in a host,usually conferred by an origin of replication, and a selection gene tofacilitate recognition of transformants are additionally incorporated.

The proteins described herein may be produced recombinantly not onlydirectly, but also as a polypeptide with a heterologous polypeptide,which is preferably a signal sequence or other polypeptide having aspecific cleavage site at the N-terminus of the mature protein orpolypeptide. The heterologous signal sequence selected preferably is onethat is recognized and processed (i.e., cleaved by a signal peptidase)by the host cell. For prokaryotic host cells that do not recognize andprocess a native signal sequence, the signal sequence is substituted bya prokaryotic signal sequence selected, for example, from the group ofthe alkaline phosphatase, penicillinase, 1pp, or heat-stable enterotoxinII leaders. For yeast secretion the native signal sequence may besubstituted by, e.g., the yeast invertase leader, a factor leader(including Saccharomyces and Kluyveromyces alpha-factor leaders), oracid phosphatase leader, the C. albicans glucoamylase leader, or thesignal described in PCT Publication No. WO 90/13646. In mammalian cellexpression, mammalian signal sequences as well as viral secretoryleaders, for example, the herpes simplex gD signal, are available. TheDNA for such precursor regions may be ligated in reading frame to DNAencoding the protein.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter).

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

A suitable selection gene for use in yeast is the trp 1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC® No. 44076or PEP4-1. Jones, Genetics, 85:12 (1977). The presence of the trp1lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC® 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the nucleicacid encoding the protein. Promoters suitable for use with prokaryotichosts include the phoA promoter, beta-lactamase and lactose promotersystems, alkaline phosphatase, a tryptophan (trp) promoter system, andhybrid promoters such as the tac promoter. However, other knownbacterial promoters are suitable. Promoters for use in bacterial systemsalso will contain a Shine-Dalgarno (S.D.) sequence operably linked tothe DNA encoding the protein.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT (SEQ ID NO: 465) region where N may be anynucleotide. At the 3′ end of most eukaryotic genes is an AATAAA (SEQ IDNO: 466) sequence that may be the signal for addition of the poly A tailto the 3′ end of the coding sequence. All of these sequences aresuitably inserted into eukaryotic expression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase or other glycolyticenzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP Patent Publication No. 73,657 and PCT Publication Nos. WO2011/124718 and WO 2012/059486. Yeast enhancers also are advantageouslyused with yeast promoters.

Transcription from vectors in mammalian host cells can be controlled,for example, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovinepapilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus,hepatitis-B virus and most preferably Simian Virus 40 (SV40), fromheterologous mammalian promoters, e.g., the ACTIN® promoter or animmunoglobulin promoter, from heat-shock promoters, provided suchpromoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature, 297:598-601 (1982) onexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the rous sarcoma virus long terminal repeat can be used as the promoter.

Transcription of a DNA encoding a protein by higher eukaryotes is oftenincreased by inserting an enhancer sequence into the vector. Manyenhancer sequences are now known from mammalian genes (globin, elastase,albumin, α-fetoprotein, and insulin). Typically, however, one will usean enhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. See alsoYaniv, Nature, 297:17-18 (1982) on enhancing elements for activation ofeukaryotic promoters. The enhancer may be spliced into the vector at aposition 5′ or 3′ to the polypeptide-encoding sequence, but ispreferably located at a site 5′ from the promoter.

Expression vectors used in eukaryotic host cells (e.g., yeast, fungi,insect, plant, animal, human, or nucleated cells from othermulticellular organisms) will also contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 5′ and, occasionally 3′,untranslated regions of eukaryotic or viral DNAs or cDNAs. These regionscontain nucleotide segments transcribed as polyadenylated fragments inthe untranslated portion of the mRNA encoding the polypeptide. Oneuseful transcription termination component is the bovine growth hormonepolyadenylation region. See WO 94/11026 and the expression vectordisclosed therein.

The recombinant DNA can also include any type of protein tag sequencethat may be useful for purifying the proteins. Examples of protein tagsinclude but are not limited to a histidine tag, a FLAG® tag, a myc tag,an HA tag, or a GST tag. Appropriate cloning and expression vectors foruse with bacterial, fungal, yeast, and mammalian cellular hosts can befound in Cloning Vectors: A Laboratory Manual, Elsevier, New York(1985), the relevant disclosure of which is hereby incorporated byreference.

The expression construct may be introduced into the host cell using amethod appropriate to the host cell, as will be apparent to one of skillin the art. A variety of methods for introducing nucleic acids into hostcells are known in the art, including, but not limited to,electroporation; transfection employing calcium chloride, rubidiumchloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; and infection (where thevector is an infectious agent).

Suitable host cells include prokaryotes, yeast, mammalian cells, orbacterial cells. Suitable bacteria include gram negative or grampositive organisms, for example, E. coli or Bacillus spp. Yeast,preferably from the Saccharomyces species, such as S. cerevisiae, mayalso be used for production of polypeptides. Various mammalian or insectcell culture systems can also be employed to express recombinantproteins. Baculovirus systems for production of heterologous proteins ininsect cells are reviewed by Luckow et al. (Bio/Technology, 6:47(1988)). Examples of suitable mammalian host cell lines includeendothelial cells, COS-7 monkey kidney cells, CV-1, L cells, C127, 3T3,Chinese hamster ovary (CHO), human embryonic kidney cells, HeLa, 293,293T, and BHK cell lines. Purified proteins are prepared by culturingsuitable host/vector systems to express the recombinant proteins. TheFBS protein is then purified from culture media or cell extracts.

V. Protein Production

Host cells are transformed with the herein-described expression orcloning vectors for protein production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

The host cells used to produce the proteins may be cultured in a varietyof media. Commercially available media such as Ham's F 10 (Sigma),Minimal Essential Medium ((MEM), (Sigma)), RPMI-1640 (Sigma), andDulbecco's Modified Eagle's Medium ((DMEM), (Sigma)) are suitable forculturing the host cells. In addition, any of the media described in Hamet al., Meth. Enzymol., 58:44 (1979), Barnes et al., Anal. Biochem.,102:255 (1980), U.S. Pat. No. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; PCT Publication Nos. WO 90/03430; WO 87/00195;or U.S. Pat. No. RE30,985 may be used as culture media for the hostcells. Any of these media may be supplemented as necessary with hormonesand/or other growth factors (such as insulin, transferrin, or epidermalgrowth factor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleotides (such as adenosine andthymidine), antibiotics (such as Gentamycin drug), trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

Proteins disclosed herein can also be produced using cell-freetranslation systems. For such purposes the nucleic acids encoding theprotein must be modified to allow in vitro transcription to produce mRNAand to allow cell-free translation of the mRNA in the particularcell-free system being utilized (eukaryotic such as a mammalian or yeastcell-free translation system or prokaryotic such as a bacterialcell-free translation system).

Proteins can also be produced by chemical synthesis (e.g., by themethods described in Solid Phase Peptide Synthesis, Second Edition, ThePierce Chemical Co., Rockford, Ill. (1984)). Modifications to theprotein can also be produced by chemical synthesis.

The proteins disclosed herein can be purified by isolation/purificationmethods for proteins generally known in the field of protein chemistry.Non-limiting examples include extraction, recrystallization, salting out(e.g., with ammonium sulfate or sodium sulfate), centrifugation,dialysis, ultrafiltration, adsorption chromatography, ion exchangechromatography, hydrophobic chromatography, normal phase chromatography,reversed-phase chromatography, gel filtration, gel permeationchromatography, affinity chromatography, electrophoresis, countercurrentdistribution or any combinations of these. After purification, proteinsmay be exchanged into different buffers and/or concentrated by any of avariety of methods known to the art, including, but not limited to,filtration and dialysis.

The purified protein is preferably at least 85% pure, more preferably atleast 95% pure, and most preferably at least 98% or 99% pure. Regardlessof the exact numerical value of the purity, the protein is sufficientlypure for use as a pharmaceutical product.

One method for expressing Adnectins in E. coli is as follows. A nucleicacid encoding an Adnectin is cloned into the PET9d vector upstream of aHIS₆tag and are transformed into E. coli BL21 DE3 plysS cells andinoculated in 5 ml LB medium containing 50 μg/mL kanamycin in a 24-wellformat and grown at 37° C. overnight. Fresh 5 ml LB medium (50 μg/mLkanamycin) cultures are prepared for inducible expression by aspirationof 200 μl from the overnight culture and dispensing it into theappropriate well. The cultures are grown at 37° C. until A₆₀₀ 0.6-0.9.After induction with 1 mM isopropyl-β-thiogalactoside (IPTG), theculture is expressed for 6 hours at 30° C. and harvested bycentrifugation for 10 minutes at 2750 g at 4° C.

Cell pellets (in 24-well format) are lysed by resuspension in 450 μl ofLysis buffer (50 mM NaH₂PO₄, 0.5 M NaCl, 1× Complete™ Protease InhibitorCocktail-EDTA free (Roche), 1 mM PMSF, 10 mM CHAPS, 40 mM imidazole, 1mg/ml lysozyme, 30 μg/ml DNAse, 2 μg/ml aprotonin, pH 8.0) and shaken atroom temperature for 1-3 hours. Lysates are cleared and re-racked into a96-well format by transfer into a 96-well Whatman GF/D Unifilter fittedwith a 96-well, 1.2 ml catch plate and filtered by positive pressure.The cleared lysates are transferred to a 96-well Nickel orCobalt-Chelating Plate that had been equilibrated with equilibrationbuffer (50 mM NaH₂PO₄, 0.5 M NaCl, 40 mM imidazole, pH 8.0) and areincubated for 5 min. Unbound material is removed by positive pressure.The resin is washed twice with 0.3 ml/well with Wash buffer #1 (50 mMNaH₂PO₄, 0.5 M NaCl, 5 mM CHAPS, 40 mM imidazole, pH 8.0). Each wash isremoved by positive pressure. Prior to elution, each well is washed with50 μl Elution buffer (PBS+20 mM EDTA), incubated for 5 min, and thiswash is discarded by positive pressure. Protein is eluted by applying anadditional 100 μl of Elution buffer to each well. After a 30 minuteincubation at room temperature, the plate(s) are centrifuged for 5minutes at 200 g and eluted protein collected in 96-well catch platescontaining 5 μl of 0.5 M MgCl₂ added to the bottom of elution catchplate prior to elution. Eluted protein is quantified using a totalprotein assay with wild-type ¹⁰Fn3 domain as the protein standard.

A method for midscale expression and purification of insoluble Adnectinsis as follows. An nucleic acid endcoding an Adnectin(s) followed by theHIS₆tag, is cloned into a pET9d (EMD Bioscience, San Diego, Calif.)vector and are expressed in E. coli HMS174 cells. Twenty ml of aninoculum culture (generated from a single plated colony) is used toinoculate 1 liter of LB medium containing 50 μg/ml carbenicillin and 34μg/ml chloramphenicol. The culture is grown at 37° C. until A₆₀₀0.6-1.0. After induction with 1 mM isopropyl-β-thiogalactoside (IPTG)the culture is grown for 4 hours at 30° C. and is harvested bycentrifugation for 30 minutes at >10,000 g at 4° C. Cell pellets arefrozen at −80° C. The cell pellet is resuspended in 25 ml of lysisbuffer (20 mM aH2PO₄, 0.5 M NaCl, 1× Complete Protease InhibitorCocktail-EDTA free (Roche), ImM PMSF, pH 7.4) using an ULTRA-TURRAX®homogenizer (IKA works) on ice. Cell lysis is achieved by high pressurehomogenization (>18,000 psi) using a Model M-1 10S MICROFLUIDIZER®(Microfluidics). The insoluble fraction is separated by centrifugationfor 30 minutes at 23,300 g at 4° C. The insoluble pellet recovered fromcentrifugation of the lysate is washed with 20 mM sodiumphosphate/500 mMNaCl, pH7.4. The pellet is resolubilized in 6.0M guanidine hydrochloridein 20 mM sodium phosphate/500M NaCl pH 7.4 with sonication followed byincubation at 37 degrees for 1-2 hours. The resolubilized pellet isfiltered to 0.45 m and loaded onto a Histrap column equilibrated withthe 20 mM sodium phosphate/500 M NaCl/6.0 M guanidine pH 7.4 buffer.After loading, the column is washed for an additional 25 CV with thesame buffer. Bound protein is eluted with 50 mM Imidazole in 20 mMsodium phosphate/500 mM NaCl/6.0 M guan-HCl pH7.4. The purified proteinis refolded by dialysis against 50 mM sodium acetate/150 mM NaCl pH 4.5.

A method for midscale expression and purification of soluble Adnectinsis as follows. A nucleic acid encoding an Adnectin(s), followed by theHIS₆tag, is cloned into a pET9d (EMD Bioscience, San Diego, Calif.)vector and expressed in E. coli HMS 174 cells. Twenty ml of an inoculumculture (generated from a single plated colony) is used to inoculate 1liter of LB medium containing 50 μg/ml carbenicillin and 34 μg/mlchloramphenicol. The culture is grown at 37° C. until A₆₀₀ 0.6-1.0.After induction with 1 mM isopropyl-β-thiogalactoside (IPTG), theculture is grown for 4 hours at 30° C. and harvested by centrifugationfor 30 minutes at >10,000 g at 4° C. Cell pellets are frozen at −80° C.The cell pellet is resuspended in 25 ml of lysis buffer (20 mM NaH₂P0₄,0.5 M NaCl, 1× Complete Protease Inhibitor Cocktail-EDTA free (Roche),ImM PMSF, pH 7.4) using an ULTRA-TURRAX® homogenizer (IKA works) on ice.Cell lysis is achieved by high pressure homogenization (>18,000 psi)using a Model M-1 10S MICROFLUIDIZER® (Microfluidics). The solublefraction is separated by centrifugation for 30 minutes at 23,300 g at 4°C. The supernatant is clarified via 0.45 m filter. The clarified lysateis loaded onto a Histrap column (GE) pre-equilibrated with the 20 mMsodium phosphate/500M NaCl pH 7.4. The column is then washed with 25column volumes of the same buffer, followed by 20 column volumes of 20mM sodium phosphate/500 M NaCl/25 mM Imidazole, pH 7.4 and then 35column volumes of 20 mM sodium phosphate/500 M NaCl/40 mM Imidazole, pH7.4. Protein is eluted with 15 column volumes of 20 mM sodiumphosphate/500 M NaCl/500 mM Imidazole, pH 7.4, fractions are pooledbased on absorbance at A₂ so and dialyzed against 1×PBS, 50 mM Tris, 150mM NaCl; pH 8.5 or 50 mM NaOAc; 150 mM NaCl; pH4.5. Any precipitate isremoved by filtering at 0.22 m.

VI. Biophysical and Biochemical Characterization

Binding of the anti-GPC3 Adnectins described herein may be assessed interms of equilibrium constants (e.g., dissociation, K_(D)) and in termsof kinetic constants (e.g., on-rate constant, k_(on) and off-rateconstant, k_(off)). An Adnectin will generally bind to a target moleculewith a K_(D) of less than 1 M, 500 nM, 100 nM, 10 nM, 1 nM, 500 pM, 200pM, or 100 pM, although higher K_(D) values may be tolerated where thek_(off) is sufficiently low or the k_(on), is sufficiently high.

In Vitro Assays for Binding Affinity

Exemplary assays for determining the binding affinity of an anti-GPC3Adnectin includes, but is not limited to, solution phase methods such asthe kinetic exclusion assay (KinExA) (Blake et al., JBC 1996;271:27677-85; Drake et al., Anal Biochem 2004; 328:35-43), surfaceplasmon resonance (SPR) with the Biacore system (Uppsala, Sweden)(Welford et al., Opt. Quant. Elect 1991; 23:1; Morton and Myszka,Methods in Enzymology 1998; 295:268) and homogeneous time resolvedfluorescence (HTRF) assays (Newton et al., J Biomol Screen 2008;13:674-82; Patel et al., Assay Drug Dev Technol 2008; 6:55-68).

In certain embodiments, biomolecular interactions can be monitored inreal time with the Biacore system, which uses SPR to detect changes inthe resonance angle of light at the surface of a thin gold film on aglass support due to changes in the refractive index of the surface upto 300 nm away. Biacore analysis generates association rate constants,dissociation rate constants, equilibrium dissociation constants, andaffinity constants. Binding affinity is obtained by assessing theassociation and dissociation rate constants using a Biacore surfaceplasmon resonance system (Biacore, Inc.). A biosensor chip is activatedfor covalent coupling of the target. The target is then diluted andinjected over the chip to obtain a signal in response units ofimmobilized material. Since the signal in resonance units (RU) isproportional to the mass of immobilized material, this represents arange of immobilized target densities on the matrix. Association anddissociation data are fit simultaneously in a global analysis to solvethe net rate expression for a 1:1 bimolecular interaction, yielding bestfit values for k_(on), k_(off) and R_(max) (maximal response atsaturation). Equilibrium dissociation constants for binding, K_(D)'s arecalculated from SPR measurements as k_(off)/k_(on).

In some embodiments, the anti-GPC3 Adnectins described herein exhibit aK_(D) in the SPR affinity assay described in Example 2 of 1 M or less,500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 150 nMor less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less,15 nM or less, 10 nM or less, 5 nM or less, or 1 nM or less.

In some embodiments, the anti-GPC3 Adnectin does not substantially bindto related proteins, for example, the anti-GPC3 Adnectin does notsubstantially bind to Glypican-1, Glypican-2, Glypican-4 or Glypican-6.

It should be understood that the assays described herein above areexemplary, and that any method known in the art for determining thebinding affinity between proteins (e.g., fluorescence based-transfer(FRET), enzyme-linked immunosorbent assay, and competitive bindingassays (e.g., radioimmunoassays) can be used to assess the bindingaffinities of the anti-GPC3 Adnectins described herein.

Cell Assays for Binding

In some embodiments, the anti-GPC3 Adnectin and conjugates thereof isinternalized into a cell expressing Glypican-3. Standard assays toevaluate polypeptide internalization are known in the art, including,for example, a HumZap internalization assay. To assess binding to tumorcells, e.g. Hep-3b or Hep-G2 (ATCC Deposit No. HB-8064 and HB-8065,respectively), cells can be obtained from publicly available sources,such as the American Type Culture Collection, and used in standardassays, such as flow cytometric analysis.

VII. Drug Conjugates

Also provided are polypeptides comprising a FBS domain, e,g., anAdnectin, conjugated to a therapeutic agent or drug moiety. In anAdnectin-drug conjugate (AdxDC), the FBS moiety (e.g., anti-GPC3Adnectin) is conjugated to a drug moiety, with the Adnectin functioningas a targeting agent for directing the AdxDC to a target cell expressingGPC3, such as a cancer cell. Once there, the drug is released, eitherinside the target cell or in its vicinity, to act as a therapeuticagent. For a review on the mechanism of action and use of drugconjugates as used with antibodies, e.g., in cancer therapy, see Schramaet al., Nature Rev. Drug Disc., 5:147 (2006).

Suitable drug moieties for use in drug conjugates include cytoxins orradiotoxins. A cytotoxin or cytotoxic agent includes any agent that isdetrimental to (e.g., kills) cells, including, antimetabolites,alkylating agents, DNA minor groove binders, DNA intercalators, DNAcrosslinkers, histone deacetylase inhibitors, nuclear export inhibitors,proteasome inhibitors, topoisomerase I or II inhibitors, heat shockprotein inhibitors, tyrosine kinase inhibitors, antibiotics, andanti-mitotic agents.

Examples of suitable agents include taxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Therapeuticagents also include, for example, antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), alkylating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine). Other preferred examples oftherapeutic cytotoxins that can be conjugated to an anti-GPC3 Adnectinof the invention include duocarmycins, calicheamicins, maytansines andauristatins, and derivatives thereof.

The Adnectin drug conjugates can be used to modify a given biologicalresponse, and the drug moiety is not to be construed as limited toclassical chemical therapeutic agents. For example, the drug moiety maybe a protein or polypeptide possessing a desired biological activity.Such proteins may include, for example, an enzymatically active toxin,or active fragment thereof, such as abrin, ricin A, pseudomonasexotoxin, or diphtheria toxin; a protein such as tumor necrosis factoror interferon-.gamma.; or, biological response modifiers such as, forexample, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”),interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor(“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or othergrowth factors.

An anti-GPC3 Adnectin can be conjugated to a therapeutic agent usinglinker technology available in the art. Examples of linker types thathave been used to conjugate a cytotoxin to an Adnectin include, but arenot limited to, hydrazones, thioethers, esters, disulfides andpeptide-containing linkers. A linker can be chosen that is, for example,susceptible to cleavage by low pH within the lysosomal compartment orsusceptible to cleavage by proteases, such as proteases preferentiallyexpressed in tumor tissue such as cathepsins (e.g., cathepsins B, C, D).Examples of cytotoxins are described, for example, in U.S. Pat. Nos.6,989,452, 7,087,600, and 7,129,261, and in PCT Application Nos.PCT/US02/17210, PCT/US2005/017804, PCT/US06/37793, PCT/US06/060050,PCT/US2006/060711, WO/2006/110476, and in U.S. Patent Application No.60/891,028, all of which are incorporated herein by reference in theirentirety.

In certain embodiments, the anti-GPC3 Adnecin and therapeutic agentpreferably are conjugated via a cleavable linker such as a peptidyl,disulfide, or hydrazone linker. More preferably, the linker is apeptidyl linker which may comprise Val-Cit, Ala-Val, Val-Ala-Val,Lys-Lys, Pro-Val-Gly-Val-Val (SEQ ID NO: 467), Ala-Asn-Val, Val-Leu-Lys,Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, or Glu. The FBS-DCs can beprepared according to methods similar to those described in U.S. Pat.Nos. 7,087,600; 6,989,452; and 7,129,261; PCT Publication Nos. WO02/096910; WO 07/038658; WO 07/051081; WO 07/059404; WO 08/083312; andWO 08/103693; U.S. Patent Publication Nos. 2006/0024317; 2006/0004081;and 2006/0247295; the disclosures of which are incorporated herein byreference.

A linker can itself be linked, e.g., covalently linked, e.g., usingmaleimide chemistry, to a cysteine of a PmXn moiety on the anti-GPC3Adnectin, wherein at least one X is a cysteine. For example, a linkercan be covalently linked to an anti-GPC3 Adnectin-PmXn, wherein at leastone X is a cysteine. For example, a linker can be linked to an anti-GPC3Adnectin-PmCn, wherein P is a proline, C is a cysteine, and m and n areintegers that are at least 1, e.g., 1-3. Ligation to a cysteine can beperformed as known in the art using maleimide chemistry (e.g.,Imperiali, B. et al., Protein Engineering: Nucleic Acids and MolecularBiology, Vol. 22, pp. 65-96, Gross, H. J., ed. (2009)). For attaching alinker to a cysteine on an anti-GPC3FBS, the linker may, e.g., comprisea maleinimido moiety, which moiety then reacts with the cysteine to forma covalent bond. In certain embodiments, the amino acids surrounding thecysteine are optimized to facilitate the chemical reaction. For example,a cysteine may be surrounded by negatively charged amino acid for afaster reaction relative to a cysteine that is surrounded by a stretchof positively charged amino acids (EP 1074563). Linkage of a drug moietyto a cysteine on an anti-GPC3 Adnectin is a site specific linkage.

For cancer treatment, the drug preferably is a cytotoxic drug thatcauses death of the targeted cancer cell. Cytotoxic drugs that can beused in anti-GPC3 FBS-DCs include, e.g., the following types ofcompounds and their analogs and derivatives:

-   -   (a) enediynes such as calicheamicin (see, e.g., Lee et al., J.        Am. Chem. Soc. 1987, 109, 3464 and 3466) and uncialamycin (see,        e.g., Davies et al., WO 2007/038868 A2 (2007); Chowdari et al.,        U.S. Pat. No. 8,709,431 B2 (2012); and Nicolaou et al., WO        2015/023879 A1 (2015));    -   (b) tubulysins (see, e.g., Domling et al., U.S. Pat. No.        7,778,814 B2 (2010); Cheng et al., U.S. Pat. No. 8,394,922 B2        (2013); and Cong et al., U.S. Pat. No. 8,980,824 B2 (2015));    -   (c) DNA alkylators such as analogs of CC-1065 and duocarmycin        (see, e.g., Boger, U.S. Pat. No. 6,5458,530 B1 (2003); Sufi et        al., U.S. Pat. No. 8,461,117 B2 (2013); and Zhang et al., U.S.        Pat. No. 8,852,599 B2 (2014));    -   (d) epothilones (see, e.g., Vite et al., US 2007/0275904        A1 (2007) and U.S. RE42930 E (2011));    -   (e) auristatins (see, e.g., Senter et al., U.S. Pat. No.        6,844,869 B2 (2005) and Doronina et al., U.S. Pat. No. 7,498,298        B2 (2009));    -   (f) pyrrolobezodiazepine (PBD) dimers (see, e.g., Howard et al.,        US 2013/0059800 A1(2013); US 2013/0028919 A1 (2013); and WO        2013/041606 A1 (2013)); and    -   (g) maytansinoids such as DM1 and DM4 (see, e.g., Chari et al.,        U.S. Pat. No. 5,208,020 (1993) and Amphlett et al., U.S. Pat.        No. 7,374,762 B2 (2008)).

The foregoing drug moiety references, in addition to disclosing the drugmoieties proper, also disclose linkers that can be used in makingdrug-linker compounds suitable for conjugating them. Particularlypertinent disclosures relating to the preparation of drug-linkercompounds are found in Chowdari et al., U.S. Pat. No. 8,709,431 B2(2012); Cheng et al., U.S. Pat. No. 8,394,922 B2 (2013); Cong et al.,U.S. Pat. No. 8,980,824 B2 (2015); Sufi et al., U.S. Pat. No. 8,461,117B2 (2013); and Zhang et al., U.S. Pat. No. 8,852,599.

Preferably, the drug moiety is a DNA alkylator, tubulysin, auristatin,pyrrolobenzodiazepine, enediyne, or maytansinoid compound, such as:

The functional group at which conjugation is effected is the amine(—NH₂) group in the case of the first five drugs above and the methylamine (—NHMe) group in the case of the last two drugs.

To conjugate a drug to an adnectin, a linker group is needed. The drugis combined with the linker to form a drug-linker compound, which isthen conjugated to the adnectin. A drug-linker compound can berepresented by formula (I)

whereinD is a drug;T is a self-immolating group;t is 0 or 1;AA^(a) and each AA^(b) are independently selected from the groupconsisting of alanine, β-alanine, γ-aminobutyric acid, arginine,asparagine, aspartic acid, γ-carboxyglutamic acid, citrulline, cysteine,glutamic acid, glutamine, glycine, histidine, isoleucine, leucine,lysine, methionine, norleucine, norvaline, ornithine, phenylalanine,proline, serine, threonine, tryptophan, tyrosine, and valine;p is 1, 2, 3, or 4;q is 2, 3, 4, 5, 6, 7, 8, 9, or 10;r is 1, 2, 3, 4, or 5; ands is 0 or 1.

In formula II, -AA^(a)-[AA^(b)]_(p)- represents a polypeptide whoselength is determined by the value of p (dipeptide if p is 1,tetrapeptide if p is 3, etc.). AA^(a) is at the carboxy terminus of thepolypeptide and its carboxyl group forms a peptide (amide) bond with anamine nitrogen of drug D (or self-immolating group T, if present).Conversely, the last AA^(b) is at the amino terminus of the polypeptideand its α-amino group forms a peptide bond with

depending on whether s is 1 or 0, respectively. Preferred polypeptides-AA^(a)-[AA^(b)]_(p)- are Val-Cit, Val-Lys, Lys-Val-Ala, Asp-Val-Ala,Val-Ala, Lys-Val-Cit, Ala-Val-Cit, Val-Gly, Val-Gln, and Asp-Val-Cit,written in the conventional N-to-C direction, as in H₂N-Val-Cit-CO₂H).More preferably, the polypeptide is Val-Cit, Val-Lys, or Val-Ala.Preferably, a polypeptide -AA^(a)-[AA^(b)]_(p)- is cleavable by anenzyme found inside the target (cancer) cell, for example a cathepsinand especially cathepsin B.

If the subscript s is 1, drug-linker (I) contains a poly(ethyleneglycol) (PEG) group, which can advantageously improve the solubility ofdrug-linker (I), facilitating conjugation to the adnectin—a step that isperformed in aqueous media. Also, a PEG group can serve as a spacerbetween the adnectin and the peptide -AA^(a)-[AA^(b)]_(p)-, so that thebulk of the adnectin does not sterically interfere with action of apeptide-cleaving enzyme.

As indicated by the subscript t equals 0 or 1, a self-immolating group Tis optionally present. A self-immolating group is one such that cleavagefrom AA^(a) or AA^(b), as the case may be, initiates a reaction sequenceresulting in the self-immolating group disbonding itself from drug D andfreeing the latter to exert its therapeutic function. When present, theself-immolating group T preferably is a p-aminobenzyl oxycarbonyl (PABC)group, whose structure is shown below, with an asterisk (*) denoting theend of the PABC bonded to an amine nitrogen of drug D and a wavy line (

) denoting the end bonded to the polypeptide -AA^(a)-[AA^(b)]_(p)-.

Another self-immolating group that can be used is a substitutedthiazole, as disclosed in Feng, U.S. Pat. No. 7,375,078 B2 (2008).

The maleimide group in formula (I) serves as a reactive functional groupfor attachment to the adnectin via a Michael addition reaction by asulfhydryl group on the adnectin, as shown below:

Alternatively, an ε-amino group in the side chain of a lysine residue ofthe adnectin can be reacted with 2-iminothiolane to introduce a freethiol (—SH) group. The thiol group can react with the maleimide group indrug-linker (I) to effect conjugation:

Conjugation by this latter method is sometimes referred to as “randomconjugation,” as the number and position of the lysine residues modifiedby the iminothiolane is difficult to predict.

In certain embodiments, the therapeutic agent in an FBS drug conjugate,e.g., AdxDC, is tubulysin or tubulysin analog. Tubulysins belong to agroup of naturally occurring antimitotic polypeptides and depsipeptidesthat includes the phomopsins, the dolastatins, and the cryptophycins(Hamel et al., Curr. Med. Chem.-Anti-Cancer Agents, 2002, 2:19-53). Thetubulysins prevent the assembly of the tubulins into microtubules,causing the affected cells to accumulate in the G₂/M phase and undergoapoptosis (Khalil et al., ChemBioChem 2006, 7:678-683).

In addition to the naturally occurring tubulysins, synthetic tubulysinanalogs with potent cytotoxic activity which are suitable for use in theFBS scaffold drug conjugate, e.g., AdxDC, provided herein have beendescribed, for example, in U.S. Pat. Nos. 8,394,922 and 8,980,824(incorporated by reference herein).

In some embodiments, therapeutic agent in the AdxDC is a synthetictubulysin analog and has a structure represented by formula (II):

To conjugate a drug moiety, e.g., having formula II, to an FBS scaffold,e.g., an anti-GPC3 FBS scaffold, a linker moiety is used which has astructure represented by formula (III):

This linker moiety comprises a valine-citrulline (Val-Cit, recited inthe conventional N-to-C direction) dipeptide, which is designed to becleaved by the intracellular enzyme cathepsin B after the AdxDC hasreached a target cancer cell and has been internalized by it, thusreleasing the therapeutic agent to exert its cytotoxic effect (Dubowchiket al., Biorg. Med. Chem Lett., 1998, 8:3341-3346; Dubowchik et atl.,Biorg. Med. Chem Lett., 1998, 8:3347-3352; Dubowchik et al.,Bioconjugate Chem., 2002, 13:855-869).

Drug (II) and linker (III) are coupled to produce a drug-linker compoundhaving a structure represented by formula (IV), which is then conjugatedto the adnectin. The preparation of drug-linker (IV) is described inCheng et al., U.S. Pat. No. 8,394,922 B2 (2013) (see, e.g., FIG. 20b andExample 22), the disclosure of which is incorporated herein. Thoseskilled in the art will appreciate that, in the instance of drug-linker(IV), neither of a optional self-immolating group or a PEG group arepresent, but that such groups can be incorporated if desired.

In the preparation of an AdxDC conjugate, a therapeutic agent-linkercompound having a structure represented by formula (IV) is prepared,which is then conjugated to the FBS scaffold.

In some embodiments, the FBS-drug conjugate has a structure representedby formula (V).

wherein m is 1, 2, 3, 4 or more. In certain embodiments, m is 1. Inother embodiments, m is 2.

In one embodiment, a thiol group in the side chain of a C-terminalcysteine residue of the anti-GPC3 Adnectin (Adx) is reacted with themaleimide group in compound (V) to effect conjugation:

In another embodiment, the thiol groups of two cysteines located at theC-terminus of the anti-GPC3 Adnectin (Adx) is reacted with the maleimidegroup in compound (IV) to effect conjugation of two drug molecules perAdnectin (e.g., DAR2, FIG. 2).

In some embodiments, the anti-GPC3Adx in the conjugate has an amino acidsequence as described above in Section IA which has been modified tocontain a C-terminal tail comprising a cysteine. In some embodiments,the anti-GPC3Adx comprises a core amino acid sequence selected from thegroup consisting of SEQ ID NOs: 5, 9-10, 18, 22-23, 31, 35-3644, 48-49,57, 61-62, 70, 74-75, 83, 87-88, 98, 102-105, 128, 130-132, 155,157-159, 180, 184-186, 20-, 211-213, 236, 238-240, 263, 265-267, 290,292-294, 317 and 319-321, which is modified to contain a C-terminalmoiety comprising a cysteine.

In some embodiments, the anti-GPC3Adx has been modified to contain aC-terminal moiety consisting P_(m)CX_(n) or P_(m)CX_(n1)CX_(n2), asdefined herein. In certain embodiments, the C-terminal moiety consistsof PC, PCC or any one of the C-terminal sequences described herein,e.g., amino acid sequences set forth in SEQ ID NOs: 409-423. In certainembodiments, the C-terminal moiety consists of PC or PCPPPPPC (SEQ IDNO: 416).

In certain embodiments, the modified anti-GPC3 Adx comprises aC-terminal moiety consisting of P_(m)CX_(n), and is selected from thegroup consisting of SEQ ID NOs: 11-14, 24-27, 37-40, 50-53, 63-66,76-79, 89-93, 106-118, 133-145, 160-172, 187-199, 214-226, 241-253,268-280, 295-307, and 322-334.

In certain embodiments, the modified anti-GPC3 Adx comprises aC-terminal moiety consisting of P_(m)CX_(n1)CX_(n2), and is selectedfrom the group consisting of SEQ ID NOs: 15-17, 28-30, 41-43, 54-57,67-70, 80-83, 94-97, 119-127, 146-154, 173-181, 200-208, 227-235,254-262, 281-289, 308-316, and 335-343.

In particular embodiments, the anti-GPC3 Adx for use in the drugconjugate is any one of SEQ ID NOs: 114-118; 123-127; 141-145; 150-154;168-172; 177-181; 195-199; 204-208; 222-226; 231-235; 249-253; 258-262;276-280; 285-289; 303-307; 312-316; 330-334; and 339-343..

Anti-GPC3 Adnectins described herein also can be conjugated to aradioactive isotope to generate cytotoxic radiopharmaceuticals. Examplesof radioactive isotopes that can be conjugated to antibodies for usediagnostically or therapeutically include, but are not limited to,iodine¹³¹, indium¹¹¹, yttrium⁹⁰ and lutetium⁷⁷. Methods for preparingradioconjugates are established in the art.

VIII. Pharmaceutical Compositions

Also provided are pharmaceutically acceptable compositions comprisingthe anti-GPC3 Adnectins and conjugates described herein, wherein thecomposition is essentially endotoxin and/or pyrogen free.

Methods well known in the art for making formulations are found, forexample, in “Remington: The Science and Practice of Pharmacy” (20th ed.,ed. A. R. Gennaro A R., 2000, Lippincott Williams & Wilkins,Philadelphia, Pa.). Formulations for parenteral administration may, forexample, contain excipients, sterile water, saline, polyalkylene glycolssuch as polyethylene glycol, oils of vegetable origin, or hydrogenatednapthalenes. Biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylenecopolymers may be used to control the release of the compounds.Nanoparticulate formulations (e.g., biodegradable nanoparticles, solidlipid nanoparticles, liposomes) may be used to control thebiodistribution of the compounds. Other potentially useful parenteraldelivery systems include ethylene-vinyl acetate copolymer particles,osmotic pumps, implantable infusion systems, and liposomes. Theconcentration of the compound in the formulation varies depending upon anumber of factors, including the dosage of the drug to be administered,and the route of administration.

Therapeutic formulations comprising proteins are prepared for storage bymixing the described proteins having the desired degree of purity withoptional physiologically acceptable carriers, excipients or stabilizers(Osol, A., ed., Remington's Pharmaceutical Sciences, 16th Edition(1980)), in the form of aqueous solutions, lyophilized or other driedformulations. Acceptable carriers, excipients, or stabilizers arenontoxic to recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyidimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrans; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as Tween, PLURONIC® or polyethylene glycol (PEG).

The polypeptide may be optionally administered as a pharmaceuticallyacceptable salt, such as non-toxic acid addition salts or metalcomplexes that are commonly used in the pharmaceutical industry.Examples of acid addition salts include organic acids such as acetic,lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic,palmitic, suberic, salicylic, tartaric, methanesulfonic,toluenesulfonic, or trifluoroacetic acids or the like; polymeric acidssuch as tannic acid, carboxymethyl cellulose, or the like; and inorganicacid such as hydrochloric acid, hydrobromic acid, sulfuric acidphosphoric acid, or the like. Metal complexes include zinc, iron, andthe like. In one example, the polypeptide is formulated in the presenceof sodium acetate to increase thermal stability.

The formulations herein may also contain more than one active compoundsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. Such molecules are suitably present in combination in amountsthat are effective for the purpose intended.

The proteins may also be entrapped in microcapsule prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsule andpoly-(methylmethacylate) microcapsule, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nanoparticles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Osol, A., ed., Remington'sPharmaceutical Sciences, 16th Edition (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the proteins described herein, whichmatrices are in the form of shaped articles, e.g., films, ormicrocapsule. Examples of sustained-release matrices include polyesters,hydrogels (for example, poly(2-hydroxyethyl -methacrylate), orpoly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and y ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT® (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated proteins remain in the body for a long time,they may denature or aggregate as a result of exposure to moisture at37° C., resulting in a loss of biological activity and possible changesin immunogenicity. Rational strategies can be devised for stabilizationdepending on the mechanism involved. For example, if the aggregationmechanism is discovered to be intermolecular S—S bond formation throughthio-disulfide interchange, stabilization may be achieved by modifyingsulfhydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, and developing specificpolymer matrix compositions.

For therapeutic applications, the proteins are administered to asubject, in a pharmaceutically acceptable dosage form. They can beadministered intravenously as a bolus or by continuous in over a periodof time, by intramuscular, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, topical, or inhalation routes. The protein may alsobe administered by intratumoral, peritumoral, intralesional, orperilesional routes, to exert local as well as systemic therapeuticeffects. Suitable pharmaceutically acceptable carriers, diluents, andexcipients are well known and can be determined by those of skill in theart as the clinical situation warrants. Examples of suitable carriers,diluents and/or excipients include: (1) Dulbecco's phosphate bufferedsaline, pH about 7.4, containing about 1 mg/ml to 25 mg/ml human serumalbumin, (2) 0.9% saline (0.9% w/v NaCl), and (3) 5% (w/v) dextrose. Themethods of the present invention can be practiced in vitro, in vivo, orex vivo.

Administration of proteins, and one or more additional therapeuticagents, whether co-administered or administered sequentially, may occuras described above for therapeutic applications. Suitablepharmaceutically acceptable carriers, diluents, and excipients forco-administration will be understood by the skilled artisan to depend onthe identity of the particular therapeutic agent being co-administered.

When present in an aqueous dosage form, rather than being lyophilized,the protein typically will be formulated at a concentration of about 0.1mg/ml to 100 mg/ml, although wide variation outside of these ranges ispermitted. For the treatment of disease, the appropriate dosage ofproteins will depend on the type of disease to be treated, the severityand course of the disease, whether the proteins are administered forpreventive or therapeutic purposes, the course of previous therapy, thepatient's clinical history and response to the fusion, and thediscretion of the attending physician. The protein is suitablyadministered to the patient at one time or over a series of treatments.

A therapeutically effective dose refers to a dose that produces thetherapeutic effects for which it is administered. The exact dose willdepend on the disorder to be treated, and may be ascertained by oneskilled in the art using known techniques. Preferred dosages can rangefrom about 10 mg/square meter to about 2000 mg/square meter, morepreferably from about 50 mg/square meter to about 1000 mg/square meter.In some embodiments, the anti-GPC3Adnectin or anti-GPC3 AdxDC isadministered at about 0.01 pg/kg to about 50 mg/kg per day, 0.01 mg/kgto about 30 mg/kg per day, or 0.1 mg/kg to about 20 mg/kg per day.

The anti-GPC3Adnectin or anti-GPC3 AdxDC may be given daily (e.g., once,twice, three times, or four times daily) or less frequently (e.g., onceevery other day, once or twice weekly, once every two weeks, once everythree weeks or monthly). In addition, as is known in the art,adjustments for age as well as the body weight, general health, sex,diet, time of administration, drug interaction, and the severity of thedisease may be necessary, and will be ascertainable with routineexperimentation by those skilled in the art.

IX. Therapeutic Methods

The anti-GPC3 Adnectins and drug conjugates thereof described herein aresuitable for use in the treatment of a cancers having tumor cellsexpressing GPC3, e.g., high levels of GPC3, e.g., relative to healthytissues. In some embodiments, the cancer is selected from the groupconsisting of liver cancer (e.g., hepatocellular carcinoma (HCC) orhepatoblastoma), melanoma, sarcoma, lung cancer (e.g., squamous lungcancer) and Wilm's tumor. Additionally, the GPC3 Adnectins describedherein are suitable for use in treating refractory or recurrentmalignancies.

As used herein, the term “subject” is intended to include human andnon-human animals. Preferred subjects include human patients havingdisorders mediated by GPC3 activity or undesirable cells expressing highlevels of GPC3. When anti-GPC3 Adnectins or drug conjugates thereof areadministered together with another agent, the two can be administered ineither order or simultaneously.

For example, the anti-GPC3 Adnectins, multispecific or bispecificmolecules and the drug conjugates thereof can be used to elicit in vivoor in vitro one or more of the following biological activities: toinhibit the growth of and/or kill a cell expressing GPC3; to mediatephagocytosis or ADCC of a cell expressing GPC3 in the presence of humaneffector cells, or to potentially modulate GPC3 activity, e.g., byblocking a GPC3 ligand from binding to GPC3.

In certain embodiments, the anti-GPC3 Adnectins or anti-GPC3 AdxDCdescribed herein are combined with an immunogenic agent, for example, apreparation of cancerous cells, purified tumor antigens (includingrecombinant proteins, peptides, and carbohydrate molecules),antigen-presenting cells such as dendritic cells bearingtumor-associated antigens, and cells transfected with genes encodingimmune stimulating cytokines (He et ah, 2004). Non-limiting examples oftumor vaccines that can be used include peptides of melanoma antigens,such as peptides of gp100, MAGE antigens, Trp-2, MARTI and/ortyrosinase, or tumor cells transfected to express the cytokine GM-CSF.GPC3 blockade may also be effectively combined with standard cancertreatments, including chemotherapeutic regimes, radiation, surgery,hormone deprivation and angiogenesis inhibitors, as well as anotherimmunotherapeutic agent (e.g., an anti-PD-1, anti-CTLA-4, and anti-LAG-3Adnectins or antibodies).

Provided herein are methods of combination therapy in which an anti-GPC3Adnectin and/or or anti-GPC3 AdxDC is administered (simultaneously orsuccessively) with one or more additional agents, e.g., small moleculedrugs, antibodies or antigen binding portions thereof, that areeffective in stimulating immune responses to thereby enhance, stimulateor upregulate immune responses in a subject.

Generally, an anti-GPC3 Adnectin or anti-GPC3 AdxDC, e.g., describedherein, can be combined with an immuno-oncology agent, e.g., (i) anagonist of a stimulatory (e.g., co-stimulatory) molecule (e.g., receptoror ligand) and/or (ii) an antagonist of an inhibitory signal or molecule(e.g., receptor or ligand) on immune cells, such as T cells, both ofwhich result in amplifying immune responses, such as antigen-specific Tcell responses. In certain aspects, an immuno-oncology agent is (i) anagonist of a stimulatory (including a co-stimulatory) molecule (e.g.,receptor or ligand) or (ii) an antagonist of an inhibitory (including aco-inhibitory) molecule (e.g., receptor or ligand) on cells involved ininnate immunity, e.g., NK cells, and wherein the immuno-oncology agentenhances innate immunity. Such immuno-oncology agents are often referredto as immune checkpoint regulators, e.g., immune checkpoint inhibitor orimmune checkpoint stimulator.

In certain embodiments, a anti-GPC3 Adnectin or anti-GPC3 AdxDC isadministered with an agent that targets a stimulatory or inhibitorymolecule that is a member of the immunoglobulin super family (IgSF). Forexample, anti-GPC3 Adnectins and or anti-GPC3 AdxDCs, e.g., describedherein, may be administered to a subject with an agent that targets amember of the IgSF family to increase an immune response. For example, aanti-GPC3 Adnectin or anti-GPC3 AdxDC may be administered with an agentthat targets (or binds specifically to) a member of the B7 family ofmembrane-bound ligands that includes B7-1, B7-2, B7-H1 (PD-L1), B7-DC(PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6 or aco-stimulatory or co-inhibitory receptor binding specifically to a B7family member.

An anti-GPC3 Adnectin or anti-GPC3 AdxDC may also be administered withan agent that targets a member of the TNF and TNFR family of molecules(ligands or receptors), such as CD40 and CD40L, OX-40, GITR, GITRL,OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137, TRAIL/Apo2-L,TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL,TWEAKR/Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTβR, LIGHT,DcR3, HVEM, VEGI/TL1A, TRAMP/DR3, EDA1, EDA2, TNFR1, Lymphotoxin α/TNFβ,TNFR2, TNFα, LTβR, Lymphotoxin a 102, FAS, FASL, RELT, DR6, TROY, andNGFR (see, e.g., Tansey (2009) Drug Discovery Today 00:1).

T cell responses can be stimulated by administering one or more of thefollowing agents:

-   -   (1) An antagonist (inhibitor or blocking agent) of a protein        that inhibits T cell activation (e.g., immune checkpoint        inhibitors), such as CTLA-4, PD-1, PD-L1, PD-L2, and LAG-3, as        described above, and any of the following proteins: TIM-3,        Galectin 9, CEACAM-1, BTLA, CD69, Galectin-1, TIGIT, CD 113,        GPR56, VISTA, B7-H3, B7-H4, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1,        and TIM-4; and/or    -   (2) An agonist of a protein that stimulates T cell activation,        such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L,        OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, DR3 and CD28H.

Exemplary agents that modulate one of the above proteins and may becombined with anti-GPC3 Adnectins and/or or anti-GPC3 AdxDCs, e.g.,those described herein, for treating cancer, include: Yervoy™(ipilimumab) or Tremelimumab (to CTLA-4), galiximab (to B7.1),BMS-936558 (to PD-1), MK-3475 (to PD-1), AMP224 (to B7DC), BMS-936559(to B7-H1), MPDL3280A (to B7-H1), MEDI-570 (to ICOS), AMG557 (to B7H2),MGA271 (to B7H3), IMP321 (to LAG-3), BMS-663513 (to CD137), PF-05082566(to CD137), CDX-1127 (to CD27), anti-OX40 (Providence Health Services),huMAbOX40L (to OX40L), Atacicept (to TACI), CP-870893 (to CD40),Lucatumumab (to CD40), Dacetuzumab (to CD40), Muromonab-CD3 (to CD3),Ipilumumab (to CTLA-4) and/or MK4166.

Anti-GPC3 Adnectins or anti-GPC3 AdxDCmay also be administered withpidilizumab (CT-011), although its specificity for PD-1 binding has beenquestioned.

Other molecules that can be combined with anti-GPC3 Adnectins oranti-GPC3 AdxDCs for the treatment of cancer include antagonists ofinhibitory receptors on NK cells or agonists of activating receptors onNK cells. For example, anti-GITR agonist antibodies can be combined withantagonists of KIR (e.g., lirilumab).

T cell activation is also regulated by soluble cytokines, and anti GPC3Adnectins or or anti-GPC3 AdxDCs may be administered to a subject, e.g.,having cancer, with antagonists of cytokines that inhibit T cellactivation or agonists of cytokines that stimulate T cell activation.

In certain embodiments, anti-GPC3 Adnectins or anti-GPC3 AdxDCs can beused in combination with (i) antagonists (or inhibitors or blockingagents) of proteins of the IgSF family or B7 family or the TNF familythat inhibit T cell activation or antagonists of cytokines that inhibitT cell activation (e.g., IL-6, IL-10, TGF-β, VEGF; “immunosuppressivecytokines”) and/or (ii) agonists of stimulatory receptors of the IgSFfamily, B7 family or the TNF family or of cytokines that stimulate Tcell activation, for stimulating an immune response, e.g., for treatingproliferative diseases, such as cancer.

Yet other agents for combination therapies include agents that inhibitor deplete macrophages or monocytes, including but not limited to CSF-1Rantagonists such as CSF-1R antagonist antibodies including RG7155(WO11/70024, WO 11/107553, WO 11/131407, WO13/87699, WO13/119716,WO13/132044) or FPA-008 (WO11/140249; WO13/69264; WO14/036357).

Anti-GPC3 Adnectins or anti-GPC3 AdxDCs may also be administered withagents that inhibit TGF-β signaling.

Additional agents that may be combined with an anti-GPC3 Adnectin and oranti-GPC3 AdxDCs include agents that enhance tumor antigen presentation,e.g., dendritic cell vaccines, GM-CSF secreting cellular vaccines, CpGoligonucleotides, and imiquimod, or therapies that enhance theimmunogenicity of tumor cells (e.g., anthracyclines).

Yet other therapies that may be combined with an anti-GPC3 Adnectin oranti-GPC3 AdxDC include therapies that deplete or block Treg cells,e.g., an agent that specifically binds to CD25.

Another therapy that may be combined with an anti-GPC3 Adnectin oranti-GPC3 AdxDC is a therapy that inhibits a metabolic enzyme such asindoleamine dioxigenase (IDO), dioxigenase, arginase, or nitric oxidesynthetase.

Another class of agents that may be used with a anti-GPC3 Adnectin oranti-GPC3 AdxDC includes agents that inhibit the formation of adenosineor inhibit the adenosine A2A receptor.

Other therapies that may be combined with a anti-GPC3 Adnectin oranti-GPC3 AdxDC for treating cancer include therapies thatreverse/prevent T cell anergy or exhaustion and therapies that triggeran innate immune activation and/or inflammation at a tumor site.

An anti-GPC3 Adnectin or anti-GPC3 AdxDC may be combined with more thanone immuno-oncology agent, and may be, e.g., combined with acombinatorial approach that targets multiple elements of the immunepathway, such as one or more of the following: a therapy that enhancestumor antigen presentation (e.g., dendritic cell vaccine, GM-CSFsecreting cellular vaccines, CpG oligonucleotides, imiquimod); a therapythat inhibits negative immune regulation e.g., by inhibiting CTLA-4and/or PD1/PD-L1/PD-L2 pathway and/or depleting or blocking Tregs orother immune suppressing cells; a therapy that stimulates positiveimmune regulation, e.g., with agonists that stimulate the CD-137, OX-40,and/or GITR pathway and/or stimulate T cell effector function; a therapythat increases systemically the frequency of anti-tumor T cells; atherapy that depletes or inhibits Tregs, such as Tregs in the tumor,e.g., using an antagonist of CD25 (e.g., daclizumab) or by ex vivoanti-CD25 bead depletion; a therapy that impacts the function ofsuppressor myeloid cells in the tumor; a therapy that enhancesimmunogenicity of tumor cells (e.g., anthracyclines); adoptive T cell orNK cell transfer including genetically modified cells, e.g., cellsmodified by chimeric antigen receptors (CAR-T therapy); a therapy thatinhibits a metabolic enzyme such as indoleamine dioxigenase (IDO),dioxigenase, arginase, or nitric oxide synthetase; a therapy thatreverses/prevents T cell anergy or exhaustion; a therapy that triggersan innate immune activation and/or inflammation at a tumor site;administration of immune stimulatory cytokines; or blocking of immunorepressive cytokines.

Anti-GPC3 Adnectins and or anti-GPC3 AdxDCs described herein can be usedtogether with one or more of agonistic agents that ligate positivecostimulatory receptors, blocking agents that attenuate signalingthrough inhibitory receptors, antagonists, and one or more agents thatincrease systemically the frequency of anti-tumor T cells, agents thatovercome distinct immune suppressive pathways within the tumormicroenvironment (e.g., block inhibitory receptor engagement (e.g.,PD-L1/PD-1 interactions), deplete or inhibit Tregs (e.g., using ananti-CD25 monoclonal antibody (e.g., daclizumab) or by ex vivo anti-CD25bead depletion), inhibit metabolic enzymes such as IDO, orreverse/prevent T cell anergy or exhaustion) and agents that triggerinnate immune activation and/or inflammation at tumor sites.

Provided herein is the use of any anti-GPC3 Adnectin described hereinfor the preparation of a medicament for treating subjects afflicted withcancer. The disclosure provides medical uses of any anti-GPC3 Adnectindescribed herein corresponding to all the embodiments of the methods oftreatment employing an anti-GPC3 Adnectin described herein.

X. Detectable Labels

The anti-GPC3 Adnectins described herein also are useful in a variety ofdiagnostic and imaging applications. In certain embodiments, ananti-GPC3 Adnectin is labeled with a moiety that is detectable in vivoand such labeled Adnectins may be used as in vivo imaging agents, e.g.,for whole body imaging. For example, in one embodiment, a method fordetecting a GPC3 positive tumor in a subject comprises administering tothe subject an anti-GPC3 Adnectin linked to a detectable label, andfollowing an appropriate time, detecting the label in the subject.

An anti-GPC3 Adnectin imaging agent may be used to diagnose a disorderor disease associated with increased levels of GPC3, for example, acancer in which a tumor selectively overexpresses GPC3. In a similarmanner, an anti-GPC3 Adnectin can be used to monitor GPC3 levels in asubject, e.g., a subject that is being treated to reduce GPC3 levelsand/or GPC3 positive cells (e.g., tumor cells). The anti-GPC3 Adnectinsmay be used with or without modification, and may be labeled by covalentor non-covalent attachment of a detectable moiety.

Detectable labels can be any of the various types used currently in thefield of in vitro diagnostics, including particulate labels includingmetal sols such as colloidal gold, isotopes such as I¹²⁵ or Tc⁹⁹presented for instance with a peptidic chelating agent of the N₂S₂, N₃Sor N₄ type, chromophores including fluorescent markers, biotin,luminescent markers, phosphorescent markers and the like, as well asenzyme labels that convert a given substrate to a detectable marker, andpolynucleotide tags that are revealed following amplification such as bypolymerase chain reaction. A biotinylated anti-GPC3 FBS would then bedetectable by avidin or streptavidin binding. Suitable enzyme labelsinclude horseradish peroxidase, alkaline phosphatase and the like. Forinstance, the label can be the enzyme alkaline phosphatase, detected bymeasuring the presence or formation of chemiluminescence followingconversion of 1,2 dioxetane substrates such as adamantyl methoxyphosphoryloxy phenyl dioxetane (AMPPD), disodium3-(4-(methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro)tricyclo{3.3.1.13,7}decan}-4-yl) phenyl phosphate (CSPD), as well as CDP and CDP-STAR®or other luminescent substrates well-known to those in the art, forexample the chelates of suitable lanthanides such as Terbium(III) andEuropium(III).

Detectable moieties that may be used include radioactive agents, suchas: radioactive heavy metals such as iron chelates, radioactive chelatesof gadolinium or manganese, positron emitters of oxygen, nitrogen, iron,carbon, or gallium, ¹⁸F ⁶⁰Cu, ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, ¹²⁴I, ⁸⁶Y, ⁸⁹Zr, ⁶⁶Ga,⁶⁷Ga, ⁶⁸Ga, ⁴⁴Sc, ⁴⁷Sc, ¹¹C, ¹¹¹In, ^(114m)In, ¹¹⁴In, ¹²⁵I, ¹²⁴I, ¹³¹I,¹²³I, ¹³¹I, ¹²³I, ³²Cl, ³³Cl, ³⁴Cl, ⁷⁴Br, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ⁷⁸Br, ⁸⁹Zr,¹⁸⁶Re, ¹⁸⁸Re, ⁸⁶Y, ⁹⁰Y, ¹⁷⁷Lu, ⁹⁹Tc, ²¹²Bi, ²¹³Bi, ²¹²Pb, ²²⁵Ac, or¹⁵³Sm.

The detection means is determined by the chosen label. Appearance of thelabel or its reaction products can be achieved using the naked eye, inthe case where the label is particulate and accumulates at appropriatelevels, or using instruments such as a spectrophotometer, a luminometer,a fluorometer, and the like, all in accordance with standard practice.

A detectable moiety may be linked to a cysteine according to methodsknown in the art. When the detectable moiety is a radioactive agent,e.g., those described further herein, the detectable moiety is linked toan FBS through a chelating agent that is reactive with cysteines, suchas a maleimide containing chelating agent, such as maleimide-NODAGA ormaleimide-DBCO. Maleimide-NODAGA or maleimide-DBCO can be reacted with acysteine on the C-terminus of an FBS (e.g., through the PmXn moiety,wherein at least one X is a cysteine), to yield FBS-NODAGA or FBS-DBCO,respectively. Any one of the following chelating agents may be usedprovided that it comprises, or can be modified to comprise, a reactivemoiety that reacts with cysteines: DFO, DOTA and its derivatives(CB-DO2A, 3p-C-DEPA, TCMC, Oxo-DO3A), TE2A, CB-TE2A, CB-TE1A1P, CB-TE2P,MM-TE2A, DM-TE2A, diamsar and derivatives, NODASA, NODAGA, NOTA, NETA,TACN-TM, DTPA, 1B4M-DTPA, CHX-A”-DTPA, TRAP (PRP9), NOPO, AAZTA andderivatives (DATA), H₂dedpa, H₄octapa, H₂azapa, H₅decapa, H₆phospa,HBED, SHBED, BPCA, CP256, PCTA, HEHA, PEPA, EDTA, TETA, and TRITA basedchelating agents, and close analogs and derivatives thereof.

In certain embodiments, an FBS is labeled with a PET tracer and used asan in vivo imaging agent. For example, an FBS may be labeled with thePET tracer ⁶⁴Cu. ⁶⁴Cu may be linked to an FBS with a C-terminal cysteinewith a chelating agent, such as maleimide-NODAGA.

Other art-recognized methods for labelling polypeptides withradionuclides such as ⁶⁴Cu and ¹⁸F for synthesizing the anti-GPC3Adnectin-based imaging agents described herein may also be used. See,e.g., US2014/0271467; Gill et al., Nature Protocols 2011; 6:1718-25;Berndt et al. Nuclear Medicine and Biology 2007; 34:5-15, Inkster etal., Bioorganic & Medicinal Chemistry Letters 2013; 23:3920-6, thecontents of which are herein incorporated by reference in theirentirety.

In certain embodiments, a GPC3 imaging agent comprises a PEG molecule(e.g., 5KDa PEG, 6KDa PEG, 7KDa PEG, 8KDa PEG, 9KDa PEG, or 10KDa PEG)to increase the blood PK of the imaging agent by small increments toenhance the imaging contrast or increase avidity of the anti-GPC3Adnectin based imaging agent.

XI. Detection of GPC3 In Vivo Detection Methods

In certain embodiments, the labeled anti-GPC3 Adnectins can be used toimage GPC3-positive cells or tissues, e.g., GPC3 expressing tumors. Forexample, the labeled anti-GPC3 Adnectin is administered to a subject inan amount sufficient to uptake the labeled Adnectin into the tissue ofinterest (e.g., the GPC3-expressing tumor). The subject is then imagedusing an imaging system such as PET for an amount of time appropriatefor the particular radionuclide being used. The labeled anti-GPC3Adnectin-bound GPC3-expressing cells or tissues, e.g., GPC3-expressingtumors, are then detected by the imaging system.

PET imaging with a GPC3 imaging agent may be used to qualitatively orquantitatively detect GPC3. A GPC3 imaging agent may be used as abiomarker, and the presence or absence of a GPC3 positive signal in asubject may be indicative that, e.g., the subject would be responsive toa given therapy, e.g., a cancer therapy, or that the subject isresponding or not to a therapy.

In certain embodiments, the progression or regression of disease (e.g.,tumor) can be imaged as a function of time or treatment. For instance,the size of the tumor can be monitored in a subject undergoing cancertherapy (e.g., chemotherapy, radiotherapy) and the extent of regressionof the tumor can be monitored in real-time based on detection of thelabeled anti-GPC3 Adnectin.

The amount effective to result in uptake of the imaging agent (e.g.,¹⁸F-Adnectin imaging agent, ⁶⁴Cu-Adnectin imaging agent) into the cellsor tissue of interest (e.g., tumors) may depend upon a variety offactors, including for example, the age, body weight, general health,sex, and diet of the host; the time of administration; the route ofadministration; the rate of excretion of the specific probe employed;the duration of the treatment; the existence of other drugs used incombination or coincidental with the specific composition employed; andother factors.

In certain embodiments, imaging of tissues expressing GPC3 is effectedbefore, during, and after administration of the labeled anti-GPC3Adnectin.

In certain embodiments, the anti-GPC3 Adnectins described herein areuseful for PET imaging of lungs, heart, kidneys, liver, and skin, andother organs, or tumors associated with these organs which express GPC3.

In certain embodiments, the anti-GPC3 imaging agents provide a contrastof at least 50%, 75%, 2, 3, 4, 5 or more. The Examples show that allanti-GPC3 Adnectins that were used provided a PET contrast of 2 or more,and that the affinity of the Adnectins was not important.

When used for imaging (e.g., PET) with short half-life radionuclides(e.g., ¹⁸F), the radiolabeled anti-GPC3 Adnectins are preferablyadministered intravenously. Other routes of administration are alsosuitable and depend on the half-life of the radionuclides used.

In certain embodiments, the anti-GPC3 imaging agents described hereinare used to detect GPC3 positive cells in a subject by administering tothe subject an anti-GPC3 imaging agent disclosed herein, and detectingthe imaging agent, the detected imaging agent defining the location ofthe GPC3 positive cells in the subject. In certain embodiments, theimaging agent is detected by positron emission tomography.

In certain embodiments, the anti-GPC3 imaging agents described hereinare used to detect GPC3 expressing tumors in a subject by administeringto the subject an anti-GPC3 imaging agent disclosed herein, anddetecting the imaging agent, the detected imaging agent defining thelocation of the tumor in the subject. In certain embodiments, theimaging agent is detected by positron emission tomography.

In certain embodiments, an image of an anti-GPC3 imaging agent describedherein is obtained by administering the imaging agent to a subject andimaging in vivo the distribution of the imaging agent by positronemission tomography.

Accordingly, provided herein are methods of obtaining a quantitativeimage of tissues or cells expressing GPC3, the method comprisingcontacting the cells or tissue with an anti-GPC3 imaging agent describedherein and detecting or quantifying the tissue expressing GPC3 usingpositron emission tomography.

Also provided herein are methods of detecting a GPC3-expressing tumorcomprising administering an imaging-effective amount of an anti-GPC3imaging agent described herein to a subject having a GPC3-expressingtumor, and detecting the radioactive emissions of said imaging agent inthe tumor using positron emission tomography, wherein the radioactiveemissions are detected in the tumor.

Also provided herein are methods of diagnosing the presence of aGPC3-expressing tumor in a subject, the method comprising

-   -   administering to a subject in need thereof an anti-GPC3 imaging        agent described herein; and    -   obtaining an radio-image of at least a portion of the subject to        detect the presence or absence of the imaging agent;    -   wherein the presence and location of the imaging agent above        background is indicative of the presence and location of the        disease.

Also provided herein are methods of monitoring the progress of ananti-tumor therapy against GPC3-expressing tumors in a subject, themethod comprising

-   -   administering to a subject in need thereof an anti-GPC3 imaging        agent described herein at a first time point and obtaining an        image of at least a portion of the subject to determine the size        of the tumor;    -   administering an anti-tumor therapy to the subject;    -   administering to the subject the imaging agent at one or more        subsequent time points and obtaining an image of at least a        portion of the subject at each time point;    -   wherein the dimension and location of the tumor at each time        point is indicative of the progress of the disease.

In Vitro Detection Methods

In addition to detecting GPC3 in vivo, anti-PDL1 Adnectins, such asthose described herein, may be used for detecting a target molecule in asample. A method may comprise contacting the sample with an anti-GPC3Adnectins described herein, wherein said contacting is carried out underconditions that allow anti-GPC3 Adnectin-target complex formation; anddetecting said complex, thereby detecting said target in said sample.Detection may be carried out using any art-recognized technique, suchas, e.g., radiography, immunological assay, fluorescence detection, massspectroscopy, or surface plasmon resonance. The sample may be from ahuman or other mammal. For diagnostic purposes, appropriate agents aredetectable labels that include radioisotopes, for whole body imaging,and radioisotopes, enzymes, fluorescent labels and other suitableantibody tags for sample testing.

The detectable labels can be any of the various types used currently inthe field of in vitro diagnostics, including particulate labelsincluding metal sols such as colloidal gold, isotopes such as I¹²⁵ orTc⁹⁹ presented for instance with a peptidic chelating agent of the N₂S₂,N₃S or N₄ type, chromophores including fluorescent markers, biotin,luminescent markers, phosphorescent markers and the like, as well asenzyme labels that convert a given substrate to a detectable marker, andpolynucleotide tags that are revealed following amplification such as bypolymerase chain reaction. A biotinylated FBS would then be detectableby avidin or streptavidin binding. Suitable enzyme labels includehorseradish peroxidase, alkaline phosphatase and the like. For instance,the label can be the enzyme alkaline phosphatase, detected by measuringthe presence or formation of chemiluminescence following conversion of1,2 dioxetane substrates such as adamantyl methoxy phosphoryloxy phenyldioxetane (AMPPD), disodium3-(4-(methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro)tricyclo{3.3.1.13,7}decan}-4-yl) phenyl phosphate (CSPD), as well as CDP and CDP-Star®or other luminescent substrates well-known to those in the art, forexample the chelates of suitable lanthanides such as Terbium(III) andEuropium(III). Other labels include those set forth above in the imagingsection. The detection means is determined by the chosen label.Appearance of the label or its reaction products can be achieved usingthe naked eye, in the case where the label is particulate andaccumulates at appropriate levels, or using instruments such as aspectrophotometer, a luminometer, a fluorimeter, and the like, all inaccordance with standard practice.

XII. Kits and Articles of Manufacture

The anti-GPC3 Adnectins and drug conjugates thereof described herein canbe provided in a kit, a packaged combination of reagents inpredetermined amounts with instructions for use in the therapeutic ordiagnostic methods described herein.

For example, in certain embodiments, an article of manufacturecontaining materials useful for the treatment or prevention of thedisorders or conditions described herein, or for use in the methods ofdetection described herein, are provided. The article of manufacturecomprises a container and a label. Suitable containers include, forexample, bottles, vials, syringes, and test tubes. The containers may beformed from a variety of materials such as glass or plastic. Thecontainer may hold a composition described herein for in vivo imaging,and may have a sterile access port (for example the container may be anintravenous solution bag or a vial having a stopper pierceable by ahypodermic injection needle). The active agent in the composition is ananti-GPC3 Adnectin or anti-GPC3 AdxDC described herein. The article ofmanufacture may further comprise a second container comprising apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use.

Exemplary Embodiments

-   1. A polypeptide comprising a fibronectin based scaffold (FBS)    comprising BC, DE and FG loops, wherein one or more of the loops are    altered relative to the corresponding loop of the wild-type FBS    domain, and wherein the polypeptide binds specifically to human    glypican-3 (GPC3) with a K_(D) or 1 μM or less.-   2. The polypeptide of embodiment 1, wherein the FBS is a fibronectin    type III (Fn3) domain.-   3. The polypeptide of embodiment 2, wherein the Fn3 domain is a    human tenth fibronectin type III (¹⁰Fn3) domain.-   4. The polypeptide of any one of the preceding embodiments, wherein    the BC loop comprises an amino acid sequence selected from the group    consisting of    -   (a) SEQ ID NOs: 6, 19, 32, 45, 58, 71, 84 or 99; and    -   (b) a BC loop with 1, 2 or 3 amino acid substitutions,        insertions or deletions relative to the BC loop of SEQ ID NOs:        6, 19, 32, 45, 58, 71, 84 or 99-   5. The polypeptide of any one of the preceding embodiments, wherein    the DE loop comprises an amino acid sequence selected from the group    consisting of    -   (a) SEQ ID NOs: 7, 20, 33, 46, 59, 72, 85 or 100; and    -   (b) a DE loop with 1, 2 or 3 amino acid substitutions,        insertions or deletions relative to the DE loop of SEQ ID NOs:        7, 20, 33, 46, 59, 72, 85 or 100.-   6. The polypeptide of any one of the preceding embodiments, wherein    the FG loop comprises an amino acid sequence selected from the group    consisting of    -   (a) SEQ ID NOs: 8, 21, 34, 47, 60, 73, 86, 101, 129, 156, 183,        210, 237, 264, 291 or 318, and    -   (b) an FG loop with 1, 2 or 3 amino acid substitutions,        insertions or deletions relative to the FG loop of SEQ ID NOs:        8, 21, 34, 47, 60, 73, 86, 101, 129, 156, 183, 210, 237, 264,        291 or 318.-   7. The polypeptide of any one of the preceding embodiments, wherein    the BC loop comprises and amino acid sequence selected from the    group consisting of SEQ ID NOs: 6, 19, 32, 45, 58, 71, 84 or 99; the    DE loop comprises an amino acid sequences selected from the group    consisting of SEQ ID NOs: 7, 20, 33, 46, 59, 72, 85 and 100; and the    FG loop comprises an amino acid sequence selected from the group    consisting of SEQ ID NOs: 8, 21, 34, 47, 60, 73, 86, 101, 129, 156,    183, 210, 237, 264, 291 and 318, respectively, and wherein,    optionally, the BC, DE and/or FG loop comprises 1, 2 or 3 amino acid    substitutions.-   8. The polypeptide of any one of the preceding embodiments, wherein    -   (a) the BC, DE and FG loops comprise SEQ ID NOs: 6, 7 and 8,        respectively;    -   (b) at least one of the BC, DE and FG loops comprises 1, 2 or 3        amino acid substitutions relative to the respective BC, DE and        FG loops of SEQ ID NOs: 6, 7 and 8;    -   (c) the BC, DE and FG loops comprise SEQ ID NOs: 19, 20 and 21,        respectively;    -   (d) at least one of the BC, DE and FG loops comprises 1, 2 or 3        amino acid substitutions relative to the respective BC, DE and        FG loops of SEQ ID NOs: 19, 20 and 22;    -   (e) the BC, DE and FG loops comprise SEQ ID NOs: 32, 33 and 34,        respectively;    -   (f) at least one of the BC, DE and FG loops comprises 1, 2 or 3        amino acid substitutions relative to the respective BC, DE and        FG loops of SEQ ID NOs: 32, 33 and 34;    -   (g) the BC, DE and FG loops comprise SEQ ID NOs: 45, 46 and 47,        respectively;    -   (h) at least one of the BC, DE and FG loops comprises 1, 2 or 3        amino acid substitutions relative to the respective BC, DE and        FG loops of SEQ ID NOs: 45, 46 and 47;    -   (i) the BC, DE and FG loops comprise SEQ ID NOs: 58, 59 and 60,        respectively;    -   (j) at least one of the BC, DE and FG loops comprises 1, 2 or 3        amino acid substitutions relative to the respective BC, DE and        FG loops of SEQ ID NOs: 58, 59 and 60;    -   (k) the BC, DE and FG loops comprise SEQ ID NOs: 71, 72 and 73,        respectively;    -   (l) at least one of the BC, DE and FG loops comprises 1, 2 or 3        amino acid substitutions relative to the respective BC, DE and        FG loops of SEQ ID NOs: 71, 72 and 73;    -   (m) the BC, DE and FG loops comprise SEQ ID NOs: 84, 85 and 86,        respectively;    -   (n) at least one of the BC, DE and FG loops comprises 1, 2 or 3        amino acid substitutions relative to the respective BC, DE and        FG loops of SEQ ID NOs: 84, 85 and 86;    -   (o) the BC, DE and FG loops comprise SEQ ID NOs: 99, 100 and        101, respectively;    -   (p) at least one of the BC, DE and FG loops comprises 1, 2 or 3        amino acid substitutions relative to the respective BC, DE and        FG loops of SEQ ID NOs: 99, 100 and 101.-   9. The polypeptide of any one of the preceding embodiments, wherein    the FBS comprises the amino acid sequence set forth in SEQ ID NO: 3,    wherein BC, DE and FG loops as represented by (X)_(v), (X)_(x), and    (X)_(z), respectively, and    -   (a) comprise amino acid sequences at least 75%, 80%, 85%, 90%,        95%, 97%, 98%, or 99% identical to the BC, DE or FG loop        sequences set forth in SEQ ID NOs: 6, 7 and 8, respectively;    -   (b) comprise BC, DE, and FG loops having the amino acid        sequences of SEQ ID NOs: 6, 7 and 8, respectively;    -   (c) comprise amino acid sequences at least 75%, 80%, 85%, 90%,        95%, 97%, 98%, or 99% identical to the BC, DE or FG loop        sequences set forth in SEQ ID NOs: 19, 20 and 21, respectively;    -   (d) comprise BC, DE, and FG loops having the amino acid        sequences of SEQ ID NOs: 19, 20 and 21, respectively;    -   (e) comprise amino acid sequences at least 75%, 80%, 85%, 90%,        95%, 97%, 98%, or 99% identical to the BC, DE or FG loop        sequences set forth in SEQ ID NOs: 32, 33 and 34, respectively;    -   (f) comprise BC, DE, and FG loops having the amino acid        sequences of SEQ ID NOs:32, 33 and 34;    -   (g) comprise amino acid sequences at least 75%, 80%, 85%, 90%,        95%, 97%, 98%, or 99% identical to the BC, DE or FG loop        sequences set forth in SEQ ID NOs: 45, 46 and 47, respectively;    -   (h) comprise BC, DE, and FG loops having the amino acid        sequences of SEQ ID NOs: 45, 46 and 47;    -   (i) comprise amino acid sequences at least 75%, 80%, 85%, 90%,        95%, 97%, 98%, or 99% identical to the BC, DE or FG loop        sequences set forth in SEQ ID NOs: 58, 59 and 60, respectively;    -   (j) comprise BC, DE, and FG loops having the amino acid        sequences of SEQ ID NOs: 58, 59 and 60;    -   (k) comprise amino acid sequences at least 75%, 80%, 85%, 90%,        95%, 97%, 98%, or 99% identical to the BC, DE or FG loop        sequences set forth in SEQ ID NOs: 71, 72 and 73, respectively;    -   (l) comprise BC, DE, and FG loops having the amino acid        sequences of SEQ ID NOs: 71, 72 and 73;    -   (m) comprise amino acid sequences at least 75%, 80%, 85%, 90%,        95%, 97%, 98%, or 99% identical to the BC, DE or FG loop        sequences set forth in SEQ ID NOs: 84, 85 and 86, respectively;    -   (n) comprise BC, DE, and FG loops having the amino acid        sequences of SEQ ID NOs: 84, 85 and 86;    -   (o) comprise amino acid sequences at least 75%, 80%, 85%, 90%,        95%, 97%, 98%, or 99% identical to the BC, DE or FG loop        sequences set forth in SEQ ID NOs: 99, 100 and 101,        respectively;    -   (p) comprise BC, DE, and FG loops having the amino acid        sequences of SEQ ID NOs: 99, 100 and 101;    -   (q) comprise BC, DE, and FG loops having the amino acid        sequences of SEQ ID NOs: 99, 100 and 129;    -   (r) comprise BC, DE, and FG loops having the amino acid        sequences of SEQ ID NOs: 99, 100 and 156;    -   (s) comprise BC, DE, and FG loops having the amino acid        sequences of SEQ ID NOs: 99, 100 and 183;    -   (t) comprise BC, DE, and FG loops having the amino acid        sequences of SEQ ID NOs: 99, 100 and 210;    -   (u) comprise BC, DE, and FG loops having the amino acid        sequences of SEQ ID NOs: 99, 100 and 237;    -   (v) comprise BC, DE, and FG loops having the amino acid        sequences of SEQ ID NOs: 99, 100 and 264;    -   (w) comprise BC, DE, and FG loops having the amino acid        sequences of SEQ ID NOs: 99, 100 and 264; or    -   (x) comprise BC, DE, and FG loops having the amino acid        sequences of SEQ ID NOs: 99, 100 and 318.-   10. The polypeptide of any one of the preceding embodiments, wherein    the FBS comprises an amino acid sequence at least 80%, 85%, 90%,    95%, 98%, 99% or 100% identical to the non-BC, DE, and FG loop    regions of SEQ ID NOs: 3, 5, 18, 31, 44, 57, 70, 83 and 98.-   11. The polypeptide of any one of the preceding embodiments, wherein    the FBS comprises an amino acid sequence at least 80%, 85%, 90%,    95%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 5, 18, 31,    44, 57, 70, 83 and 98.-   12. The polypeptide of any one of the preceding embodiments, wherein    the FBS comprises an amino acid sequence at least 90%, 95%, 98%, 99%    or 100% to the amino acid sequence of any one of SEQ ID NOs 5, 9-18,    22-31, 35-44, 48-57, 61-70, 74-83, 87-98, 102-128, 130-155, 157-182,    184-209, 211-236, 238-263, 265-290, 292-317 or 319-343.-   13. The polypeptide of claim any one of the preceding embodiments,    wherein the FBS comprises an amino acid that is at least 95%    identical to the amino acid sequence of SEQ ID NOs: 98, 102-128,    129-155, 157-182, 184-209, 211-236, 238-263, 265-290, 292-317 and    319-343.-   14. The polypeptide any one of the preceding embodiments, wherein    the FBS comprises an amino acid sequence selected from the group    consisting of SEQ ID NOs: 5, 18, 31, 44, 57, 70, 83, 98, 128, 155,    182, 209, 209, 236, 263, 290 and 317.-   15. The polypeptide any one of the preceding embodiments, wherein    the FBS comprises an amino acid sequence selected from the group    consisting of SEQ ID NOs: 5, 9-18, 22-31, 35-44, 48-57, 61-70,    74-83, 87-98, 102-128, 130-155, 157-182, 184-209, 211-236, 238-263,    265-290, 292-317 and 319-343.-   16. The polypeptide of anyone of the preceding embodiments, wherein    the FBS comprises an amino acid sequence selected from the group    consisting of SEQ ID NOs: 98, 102-128, 129-155, 157-182, 184-209,    211-236, 238-263, 265-290, 292-317 and 319-343.-   17. The polypeptide of any one of the preceding embodiments, wherein    the FBS competes with an FBS comprising the amino acid sequence of    SEQ ID NO: 98 for binding with glypican-3.-   18. The polypeptide of any one of the preceding embodiments, wherein    the FBS binds to a region of 10-20 amino acid residues within    glypican-3 which comprise HQVSFF (SEQ ID NO:209).-   19. The polypeptide of any one of the preceding embodiments, wherein    the FBS binds to a region of 10-20 amino acid residues within    glypican-3 which comprise EQLLQSASM (SEQ ID NO:210).-   20. The polypeptide of any one of the preceding embodiments, wherein    the FBS binds to a discontinuous Adnectin site within glypican-3    comprising HQVSFF (SEQ ID NO:209) and EQLLQSASM (SEQ ID NO:210).-   21. The polypeptide of any one of the preceding embodiments, further    comprising a heterologous protein.-   22. The polypeptide of any one of the preceding embodiments, further    comprising one or more pharmacokinetic (PK) moieties selected from    the group consisting of polyethylene glycol, sialic acid, Fc, Fc    fragment, transferrin, serum albumin, a serum albumin binding    protein, and a serum immunoglobulin binding protein.-   23. The polypeptide of any one of the preceding embodiments, wherein    the C-terminus of the FBS is linked to a moiety consisting of the    amino acid sequence P_(m)X_(n), wherein P is proline, X is any amino    acid, m is an integer that is at least 1 and n is 0 or an integer    that is at least 1.-   24. The polypeptide of embodiment 23, wherein m is 1 or 2, and n is    an integer from 1-10.-   25. The polypeptide of embodiment 23 or 24, wherein moiety comprises    cysteine.-   26. The polypeptide of embodiment 25, wherein the moiety consists of    the amino acid sequence P_(m)CX_(n), wherein C is a cysteine, each X    is independently any amino acid.-   27. The polypeptide of embodiment 25, wherein the moiety consists of    the amino acid sequence P_(m)CX_(n1)CX_(n2), wherein each X is    independently any amino acid, n₁ and n₂ are independently 0 or an    integer that is at least 1.-   28. The polypeptide of embodiment 28, wherein n₁ and n₂ are    independently an integer from 1-5.-   29. The polypeptide of embodiment 23, wherein the moiety is selected    from the group consisting of PI, PC, PID, PIE, PIDK (SEQ ID NO:    382), PIEK (SEQ ID NO: 383), PIDKP (SEQ ID NO: 384), PIEKP (SEQ ID    NO: 385), PIDKPS (SEQ ID NO: 386), PIEKPS (SEQ ID NO: 387), PIDKPC    (SEQ ID NO: 388), PIEKPC (SEQ ID NO: 389), PIDKPSQ (SEQ ID NO: 390),    PIEKPSQ (SEQ ID NO: 391), PIDKPCQ (SEQ ID NO: 392), PIEKPCQ (SEQ ID    NO: 393), PHHHHHH (SEQ ID NO: 394) and PCHHHHHH (SEQ ID NO: 395).-   30. The polypeptide of embodiment 26, wherein the moiety is PC or    PPC.-   31. The polypeptide of embodiment 27, wherein the moiety is selected    from the group consisting of PCGC (SEQ ID NO: 412), PCPC (SEQ ID NO:    413), PCGSGC (SEQ ID NO: 414), PCPPPC (SEQ ID NO: 415), PCPPPPPC(SEQ    ID NO: 416), PCGSGSGC (SEQ ID NO: 417), PCHHHHHC (SEQ ID NO: 418),    PCCHHHHHH (SEQ ID NO: 419), PCGCHHHHHH (SEQ ID NO: 420), PCPCHHHHHH    (SEQ ID NO: 421), PCGSGCHHHHHH (SEQ ID NO: 422), PCPPPCHHHHHH (SEQ    ID NO: 423), PCPPPPPHHHHHH (SEQ ID NO: 424) and PCGSGSGCHHHHHH (SEQ    ID NO: 425).-   32. The polypeptide of embodiment 31, wherein the moiety is PCPPPPPC    (SEQ ID NO: 416).-   33. The polypeptide of any one of embodiments 25-32, wherein the    cysteine in the C-terminal moiety is conjugated to a heterologous    moiety.-   34. The polypeptide of embodiment 33, wherein the heterologous    moiety is a detectable moiety.-   35. The polypeptide of embodiment 33, wherein the heterologous    moiety is a drug moiety, and the FBS and drug moiety form a FBS-drug    conjugate.-   36. An FBS-drug conjugate comprising the FBS moiety of any one of    embodiments 1-31, wherein the drug moiety is conjugated to the FBS    moiety by a linker.-   37. The FBS-drug conjugate of embodiment 36, wherein the linker is a    hydrazones, thioether, ester, disulfide or peptide-containing    linker.-   38. The FBS-drug conjugate of embodiment 37, wherein the linker is a    peptidyl linker.-   39. The FBS-drug conjugate of embodiment 38, wherein the peptidyl    linker is Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys,    Pro-Val-Gly-Val-Val (SEQ ID NO: 467), Ala-Asn-Val, Val-Leu-Lys,    Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, or Glu.-   40. The FBS-drug conjugate of any one of embodiments 36-39, wherein    the drug moiety is a cytotoxic drug.-   41. The FBS-drug conjugate of embodiment 40, wherein the cytotoxic    drug selected from the group consisting of:    -   (a) enediynes such as calicheamicin and uncialamycin;    -   (b) tubulysins;    -   (c) DNA alkylators such as analogs of CC-1065 and duocarmycin;    -   (d) epothilones;    -   (e) auristatins;    -   (f) pyrrolobezodiazepine (PBD) dimers;    -   (g) maytansinoids such as DM1 and DM4 and analogs and        derivatives thereof.-   42. The FBS-drug conjugate of embodiment 41, wherein the drug moiety    is:

-   43. The FBS-drug conjugate of any one of embodiments 36-41, wherein    the drug moiety is a synthetic tubulysin analog having the structure    of formula (II):

-   44. The FBS-drug conjugate of any one of embodiments 36-43, wherein    the FBS and drug moiety are conjugated with a linker moiety having    the structure of formula (III):

-   45. The FBS-drug conjugate of embodiment 44, wherein the    drug-moiety-linker has the structure of formula (IV):

-   -   wherein the maleimide group is reacted with a sulfhydryl group        of a cysteine of the FBS, thereby forming a thioether bond        between the drug-moiety-linker and the FBS.

-   46. The FBS-drug conjugate of any one of embodiments 36-45, wherein    the FBS moiety comprises a C-terminal moiety comprising a cysteine.

-   47. The FBS-drug conjugate of embodiment 46, wherein the C-terminal    moiety consists of the amino acid sequence P_(m)CX_(n), wherein C is    a cysteine, each X is independently any amino acid, m is an integer    that is at least 1 and n is 0 or an integer that is at least 1.

-   48. The FBS-drug conjugate of embodiment 47, wherein m is 1 or 2,    and n is an integer from 1-10.

-   49. The FBS-drug conjugate of embodiment 46, wherein the moiety    consists of the amino acid sequence P_(m)CX_(n1)CX_(n2), wherein    each X is independently any amino acid, n₁ and n₂ are independently    0 or an integer that is at least 1.

-   50. The FBS-drug conjugate of embodiment 49, wherein n₁ and n₂ are    independently an integer from 1-5.

-   51. The FBS-drug conjugate of embodiment 47, wherein the C-terminal    moiety is PC or PPC.

-   52. The FBS-drug conjugate of embodiment 49, wherein the moiety is    selected from the group consisting of PCGC (SEQ ID NO: 412), PCPC    (SEQ ID NO: 413), PCGSGC (SEQ ID NO: 414), PCPPPC (SEQ ID NO: 415),    PCPPPPPC(SEQ ID NO: 416), PCGSGSGC (SEQ ID NO: 417), PCHHHHHC (SEQ    ID NO: 418), PCCHHHHHH (SEQ ID NO: 419), PCGCHHHHHH (SEQ ID NO:    420), PCPCHHHHHH (SEQ ID NO: 421), PCGSGCHHHHHH (SEQ ID NO: 422),    PCPPPCHHHHHH (SEQ ID NO: 423), PCPPPPPHHHHHH (SEQ ID NO: 424) and    PCGSGSGCHHHHHH (SEQ ID NO: 425).

-   53. The FBS-drug conjugate of embodiment 52, wherein the moiety is    PCPPPPPC (SEQ ID NO: 16).

-   54. An FBS-drug conjugate, having a structure represented by formula    (I)

-   -   wherein m is 1, 2, 3 or 4 and Adx is an Adnectin that binds        specifically to human GPC3 with a K_(D) or 1 μM or less, and        wherein the sulfur atom that is linked to “Adx” is a sulfur atom        of a sulfhydryl group of a cysteine of the Adnectin.

-   55. The FBS-drug conjugate of embodiment 54, wherein Adx is a human    ¹⁰Fn3 domain wherein,    -   (a) the BC, DE and FG loops comprise SEQ ID NOs: 6, 7 and 8,        respectively;    -   (b) the BC, DE and FG loops comprise SEQ ID NOs: 19, 20 and 21,        respectively;    -   (c) the BC, DE and FG loops comprise SEQ ID NOs: 32, 33 and 34,        respectively;    -   (d) the BC, DE and FG loops comprise SEQ ID NOs: 45, 46 and 47,        respectively;    -   (e) the BC, DE and FG loops comprise SEQ ID NOs: 58, 59 and 60,        respectively;    -   (f) the BC, DE and FG loops comprise SEQ ID NOs: 71, 72 and 73,        respectively;    -   (g) the BC, DE and FG loops comprise SEQ ID NOs: 84, 85 and 86,        respectively;    -   (h) the BC, DE and FG loops comprise SEQ ID NOs: 99, 100 and        101, respectively;    -   (i) the BC, DE and FG loops comprise SEQ ID NOs: 99, 100 and        129, respectively;    -   (j) the BC, DE and FG loops comprise SEQ ID NOs: 99, 100 and        156, respectively;    -   (k) the BC, DE and FG loops comprise SEQ ID NOs: 99, 100 and        183, respectively;    -   (l) the BC, DE and FG loops comprise SEQ ID NOs: 99, 100 and        210, respectively;    -   (m) the BC, DE and FG loops comprise SEQ ID NOs: 99, 100 and        237, respectively;    -   (n) the BC, DE and FG loops comprise SEQ ID NOs: 99, 100 and        264, respectively;    -   (o) the BC, DE and FG loops comprise SEQ ID NOs: 99, 100 and        264, respectively; or    -   (p) the BC, DE and FG loops comprise SEQ ID NOs: 99, 100 and        318, respectively,        and, the ¹⁰Fn3 domain comprises a C-terminal moiety consisting        of the amino acid sequence P_(m)CX_(n), or P_(m)CX_(n1)CX_(n2),        wherein each X is independently any amino acid, n₁ and n₂ are        independently 0 or an integer that is at least 1.

-   56. The FBS-drug conjugate of embodiment 55, wherein the C-terminal    moiety is PC or PPC, PCGC (SEQ ID NO: 412), PCPC (SEQ ID NO: 413),    PCGSGC (SEQ ID NO: 414), PCPPPC (SEQ ID NO: 415), PCPPPPPC(SEQ ID    NO: 416), PCGSGSGC (SEQ ID NO: 417), PCHHHHHC (SEQ ID NO: 418),    PCCHHHHHH (SEQ ID NO: 419), PCGCHHHHHH (SEQ ID NO: 420), PCPCHHHHHH    (SEQ ID NO: 421), PCGSGCHHHHHH (SEQ ID NO: 422), PCPPPCHHHHHH (SEQ    ID NO: 423), PCPPPPPHHHHHH (SEQ ID NO: 424) or PCGSGSGCHHHHHH (SEQ    ID NO: 425).

-   57. The FBS-drug conjugate of embodiment 52, wherein the moiety is    PC or PCPPPPPC (SEQ ID NO: 16).

-   58. The FBS-drug conjugate of embodiment 54, wherein Adx comprises    an amino acid sequence selected from the group consisting of SEQ ID    NOs: 12-17, 24-30, 38-43, 51-56, 64-69, 77-82, 90-97, 110-127,    137-154, 164-181, 191-208, 218-235, 245-262, 272-289, 299-316 and    326-343.

-   59. The FBS-drug conjugate of embodiment 54, wherein Adx comprises    an amino acid sequence selected from the group consisting of SEQ ID    NOs: 110-127, 137-154, 164-181, 191-208, 218-235, 245-262, 272-289,    299-316 and 326-343.

-   60. The FBS-conjugate of embodiment 54, wherein Adx comprises an    amino acid sequence selected from the group consisting of SEQ ID    NOs: 110-127.

-   61. A pharmaceutical composition comprising a polypeptide of any one    of embodiments 1-35, and a pharmaceutically acceptable carrier.

-   62. A pharmaceutical composition comprising an FBS-drug conjugate of    any one of embodiments 36-60.

-   63. The composition of embodiment 61 or 62, wherein the composition    is essentially endotoxin-free.

-   64. An isolated nucleic acid molecule encoding the polypeptide of    any one of embodiments 1-35.

-   65. An expression vector comprising a nucleotide sequence encoding    the polypeptide of any one of claims 1-35.

-   66. A cell comprising a nucleic acid encoding the polypeptide of any    one of embodiments 1-35.

-   67. A method of producing the polypeptide of any one of embodiments    1-35, comprising culturing the cell of claim 66 under conditions    suitable for expressing the polypeptide, and purifying the    polypeptide.

-   68. A method of attenuating or inhibiting a glypican-3 disease or    disorder in a subject comprising administering to the subject an    effective amount of the pharmaceutical composition of embodiment 61    or 62.

-   69. The method of embodiment 68, wherein the glypican-3 disease or    disorder is cancer.

-   70. The method of embodiment 69, wherein the cancer is    hepatocellular carcinoma, melanoma, Wilm's tumor, hepatoblastoma    ovarian cancer or squamous lung cancer.

-   71. A kit comprising the polypeptide, FBS-drug conjugate or    pharmaceutical composition of any one of embodiments 1-62, and    instructions for use.

-   72. A method of detecting or measuring glypican-3 in a sample    comprising contacting the sample with the polypeptide of any one of    embodiments 1-35, and detecting or measuring binding of the FBS to    glypican-3.

-   73. An FBS-drug conjugate, having a structure represented by formula    (I)

-   -   wherein m is 1 and Adx is an Adnectin comprising the amino acid        sequence of any one of SEQ ID NO: 110-117 and 272-289; and        wherein the sulfur atom that is linked to “Adx” is a sulfur atom        of a sulfhydryl group of the C-terminal cysteine of the        Adnectin.

-   74. An FBS-drug conjugate, having a structure represented by formula    (I)

-   -   wherein m is 2 and Adx is an Adnectin comprising the amino acid        sequence of SEQ ID NO: 119-126 and 281-288; and wherein the        sulfur atom that is linked to “Adx” is a sulfur atom of a        sulfhydryl group of each of the two C-terminal cysteines of the        Adnectin.

-   75. An FBS-drug conjugate, having a structure represented by formula    (VI)

wherein the sulfur atom linked to the cysteine is the sulfur atom of thesulfhydryl group of the cysteine.

-   76. An FBS-drug conjugate, having a structure represented by formula    (VII):

wherein the sulfur atom linked to the cysteines is the sulfur atom ofthe sulfhydryl group of the cysteines.

INCORPORATION BY REFERENCE

The contents of all figures and all references, Genbank sequences,websites, patents and published patent applications cited throughoutthis application are expressly incorporated herein by reference to thesame extent as if there were written in this document in full or inpart. The content of PCT/US2015/021466 is specifically incorporated byreference herein.

The invention is now described by reference to the following examples,which are illustrative only, and are not intended to limit the presentinvention. While the invention has been described in detail and withreference to specific embodiments thereof, it will be apparent to one ofskill in the art that various changes and modifications can be madethereto without departing from the spirit and scope thereof.

Examples Example 1: Selection, Expression and Purification ofAnti-Glypican-3 Binding Adnectins

Glypican-3 (GPC3) binding Adnectins were isolated from an Adnectinlibrary screened with a Glypican-3 protein, or were affinity matured byPROfusion from clones identified in the library. For a detaileddescription of the RNA-protein technology and fibronectin-based scaffoldprotein library screening methods see Szostak et al., U.S. Pat. Nos.6,258,558; 6,261,804; 6,214,553; 6,281,344; 6,207,446; 6,518,018; PCTPublication Nos. WO 00/34784; WO 01/64942; WO 02/032925; and Roberts etal., Proc Natl. Acad. Sci., 94:12297-12302 (1997), herein incorporatedby reference.

The amino acid and nucleotide sequences of 7 adnectins with good bindingand biophysical properties are provided below:

ADX_4578_F03 (SEQ ID NO: 10)MGVSDVPRDLEVVAATPTSLLISWHPPHPNIVSYHIYYGETGGNSPVQEFTVEGSKSTAKISGLKPGVDYTITVYAVAPEIEKYYQIWINYRTEGSGS* (SEQ ID NO: 452)ATGGGAGTTTCTGATGTGCCGCGCGACTTGGAAGTGGTTGCCGCCACCCCCACCAGCCTGCTGATCTCTTGGCATCCGCCGCATCCGAACATCGTTTCTTACCATATCTACTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGGAAGGTTCTAAATCTACTGCTAAAATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTACGCTGTTGCTCCGGAAATCGAAAAATACTACCAGATTTGGATTAATTACCGCACAGAAGGCAGCGGTTCCTAA ADX_4578_H08(SEQ ID NO: 23) MGVSDVPRDLEVVAATPTSLLISWSGYDYGDSYYRITYGETGGNSPVQEFTVPDGSNTATISGLKPGVDYTITVYAVEAYGKGYTRYTPISINYRTEIDK PSQ* (SEQ ID NO: 452)ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATCAGCTGGTCTGGTTACGACTACGGTGACTCTTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGACGGTTCTAACACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAAGCTTACGGTAAAGGTTACACTCGTTACACTCCAATTTCCATTAATTACCGCACAGAAATTGACAAA CCATCCCAGTAAADX_4578_B06 (SEQ ID NO: 36)MGVSDVPRDLEVVAATPTSLLISWFPDRYVYYITYGETGGNSPVQEFTVEGHKQTAYISGLKPGVDYTITVYAIYYYPDDFQGYPQPISINYRTEGSGS* (SEQ ID NO: 454)ATGGGAGTTTCTGATGTGCCGCGCGACTTGGAAGTGGTTGCCGCCACCCCCACCAGCCTGCTGATCTCTTGGTTCCCGGACCGTTACGTTTACTACATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGGAAGGTCATAAACAGACTGCTTACATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTACGCTATCTACTACTACCCGGACGACTTCCAGGGTTACCCGCAGCCGATTTCTATTAATTACCGCACAGAAGGCAGCGGTTCCTAA ADX_4606_F06(SEQ ID NO: 49) MGVSDVPRDLEVVAATPTSLLISWNSGHSGQYYRITYGETGGNSPVQEFTVPRYGYTATISGLKPGVDYTITVYAVAHSEASAPISINYRTEIDKPSQ*  (SEQ ID NO: 455)ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATCAGCTGGAACTCTGGTCATTCTGGTCAGTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTCGTTACGGTTACACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGCTCATTCTGAAGCTTCTGCTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGTAA ADX_5273_C01(SEQ ID NO: 62) MGVSDVPRDLEVVAATPTSLLISWSDPYEEERYYRITYGETGGNSPVQEFTVPAFHTTATISGLKPGVDYTITVYAVTYKHKYAYYYPPISINYRTEIDK PSQ* (SEQ ID NO: 456)ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATCAGCTGGTCTGACCCGTACGAAGAAGAACGATATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGCTTTCCATACTACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTTACAAACATAAATACGCTTACTACTACCCGCCAATTTCCATTAATTACCGCACAGAAATTGACAAA CCATCCCAGTAAADX_5273_D01 (SEQ ID NO: 75)MGVSDVPRDLEVVAATPTSLLISWEPSYKDDRYYRITYGETGGNSPVQEFTVPSFHQTATISGLKPGVDYTITVYAVTYEPDEYYFYYPISINYRTEIDK PSQ* (SEQ ID NO: 457)ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATCAGCTGGGAACCGTCTTACAAAGACGACCGATATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTTCTTTCCATCAGACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTTACGAACCGGACGAATACTACTTCTACTACCCAATTTCCATTAATTACCGCACAGAAATTGACAAA CCATCCCAGTAAADX_5274_ (SEQ ID NO: 88)MGVSDVPRDLEVVAATPTSLLISWSGDYHPHRYYRITYGETGGNSPVQEFTVPGEHETATISGLKPGVDYTITVYAVTYDGEKADKYPPISINYRTEIDK PSQ* (SEQ ID NO: 458)ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATCAGCTGGTCTGGTGACTACCATCCGCATCGATATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGGTGAACATGAAACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTTACGACGGTGAAAAAGCTGACAAATACCCGCCAATTTCCATTAATTACCGCACAGAAATTGACAAA CCATCCCAGTAA

The binding characteristics of the 7 GPC3 binding Adnectins weredetermined via ELISA using recombinant GPC3 and flow cytometry, usingthe GPC3 positive CHO cell line and the HepG2 human tumor cell line. Forthe flow cytometry experiments, CHO-K1 or CHO-Glypican-3 cells or HepG2tumor cell line were treated with Versene and resuspended in FACS Buffer(PBS 2.5% FBS). Adnectins diluted in FACS buffer were incubated withcells for 1 hour at 4° C. After 1 wash in FACS buffer the cells wereincubated with an anti-His antibody at 2 ug/ml and incubated for 1 hourat 4° C. After 2 washes in FACS Buffer the cells were resuspended in FIXBuffer (2.5% formaldehyde in PBS). Analysis was done with the BBBiosciences FACS Canto.

The results of the ELISA experiments are shown in Table 3, and exemplaryflow cytometry results are shown in FIG. 3A-D. The aggregation score ofthe Adnectins, as determined by Size Exclusion Chromatograph (SEC) isalso provided in the Table 2. None of these Adnectins aggregatedsignificantly.

TABLE 2 ELISA and SEC scores of human GPC3 binding Adnectins Clone ELISAOD SEC score ADX_4578_F03 0.60 2 ADX_4578_H08 1.02 2 ADX_4578_B06 0.17 3ADX_4606_F06 1.73 3 ADX_5273_C01 1.35 3 ADX_5273_D01 2.1 2 ADX_5274_E012.37 1

The C-terminus of ADX_5274_E01 was modified by inclusion of a C-terminalcysteine and a 6×His tail, to produce Adnectin ADX_6561_A01:MGVSDVPRDLEVVAATPTSLLISWSGDYHPHRYYRITYGETGGNSPVQEFTVPGEHETATISGLKPGVDYTITVYAVTYDGEKADKYPPISINYRTPCHHHHHH (SEQ ID NO: 94)

The nucleic acid encoding ADX_6561_A01 was diversified by introducing asmall fraction of substitutions at each nucleotide position that encodedan amino acid residue in loop BC, DE or FG. The resulting library ofAdnectin sequences related to ADX_6561_A01 was then subjected to invitro selection by PROfusion (mRNA display) for binding to human GPC3under high stringency conditions. The clones enriched after selectionwas completed were sequenced, expressed in HTPP format, and furtheranalyzed.

The selection identified Adnectin ADX_6077_F02 as binding to human GPC3with high affinity. The amino acid sequence of ADX_6077_F02, and thenucleotide sequence encoding it are as follows:

MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDGEKAATDWSISINYRTPCHHHHHH (SEQ ID NO: 118; the BC, DE and FG loops are shown in bold)(SEQ ID NO: 459) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATCAGCTGGTCTGATGACTACCATGCGCATCGATATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGGTGAACATGTGACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTTACGACGGTGAAAAGGCTGCCACAGATTGGTCAATTTCCATTAATTACCGCACACCGTGCCACCAT CACCACCACCACTGA

Binding of the anti-GPC3 Adnectin to other glypican molecules wastested, and the results, indicate that ADX_6077_F02 binds specificallyto human GPC3, and does not cross-react to the other human glypicans GPC1, GPC2, GPC5 and GPC6.

Example 2: Adnectins with C-Terminal Cysteines for Conjugation of a DrugMoiety

For preparing Adnectins linked to a drug moiety, the Adnectins weremodified at their C-terminus to comprise one of the following C-terminalamino acid sequences: NYRTPC (SEQ ID NO: 466; for forming DAR1Adnectins, i.e., Adnectins with a single cysteine in the linker, forlinking to a single drug moiety); NYRTPCC (SEQ ID NO: 467; for formingDAR2 Adnectins, i.e., Adnectins with two cysteines in the linker, forlinking to two drug moieties, one per cysteine); NYRTPCHHHHHH (SEQ IDNO: 468; for forming DAR1 Adnectins with a 6×His tail) andNYRTPCPPPPPCHHHHHH (SEQ NO: 469; for forming DAR2 Adnectins with a 6×Histail).

To prevent dislufide-linked dimers of the uncongugated Adnectinscontaining one or more Cysteine residues, the cysteine residue(s) of theAdnectins were carboxymethylated as follows: An Adnectin solution wastreated with a reducing agent (5 mM DTT or 5 mM TCEP) and incubated for30 minutes at room temperature. Ioodoaceamide (500 mM, Sigma P/NA3221-10VL) was added to a final concentration of 50 mM. Samples wereincubated for 1 hour in the dark at room temperature. Samples were thendialyzed to PBS or Sodium Acetate buffer.

Example 3: Production of GPC3-Adnectin Drug Conjugates (GPC3-AdxDC)

Production of Adnectins, e.g., GPC3 Adnectins:

A nucleic acid encoding an Adnectin, e.g., (SEQ ID NO: 459), whichencodes a protein having the amino acid sequenceMGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDGEKAATDWSISINYRTPCHHHHHH (SEQ ID NO: 118;ADX_6077_F02), was cloned into a pET9d (EMD Biosciences, San Diego,Calif.) vector and expressed in E. coli BL21 DE3 pLys-S cells. Twenty mlof an inoculum culture (generated from a single plated colony) was usedto inoculate 1 liter of Magic Media E. coli expression medium(Invitrogen, Catalog K6803A/B) containing 50 ug/ml Kanamycin in a 2.5Liter Ultra Yield flask (Thomson Instruments Co. P/N 931136-B). Theculture was grown at 37° C. for 6 hours, followed by 20° C. for 18 hourswith shaking at 225 RPM. After the incubation period, the culture washarvested by centrifugation for 30 minutes at >10,000 g at 4° C. Cellpellets were frozen at −80° C. The cell pellet was thawed andresuspended in 25 mL of lysis buffer (20 mM Sodium Phosphate, 500 mMSodium Chloride, 5 mM Dithiothreitol, 1× Complete™ Protease InhibitorCocktail-EDTA free (Roche) using an Ultra-Turrax homgenizer (IKA—Works)on ice. Cell lysis was achieved by high pressure homogenization (>18,000psi) using a Model M-110P Microfluidizer (Microfluidics). The insolublefraction was separated by centrifugation for 30 minutes at 23,300 g at4° C. and discarded. The soluble fraction was filtered with a 0.2 micronvacuum filter. The filtered supernatant was loaded onto a Histrap column(GE Healthcare P/N 17-5248-02) equilibrated with 20 mM SodiumPhosphate/500 mM Sodium chloride pH 7.4+5 mM DTT buffer. After loading,the column was washed with 10 CV equilibration buffer, followed by 10 CVof 40 mM Imidazole in equilibration buffer, followed by 10 CV 2.0MSodium Chloride in PBS. Bound protein was eluted with 500 mM Imidazolein 20 mM Sodium Phosphate/500 mM Sodium Chloride pH 7.4+5 mM DTT. Theeluate from the HisTrap column was buffer exchanged to 50 mM SodiumAcetate/10 mM Sodium Chloride pH 5.5 using G25 gel filtrationchromatography. The sample was then applied to a cation exchangechromatography column (SP HP, GE Healthcare 17-1152-01). Bound proteinwas eluted in a gradient of increasing sodium chloride concentration in50 mM Sodium Acetate pH 5.5 buffer. Fractions were pooled forconjugation with tubulsyin.

Production of Tubulysin Analog-Linker:

A tubulysin analog-linker compound having the structure of formula (IV)was produced as described in U.S. Pat. No. 8,394,922 (herebyincorporated by reference).

Adnectin-Drug Conjugation:

Conjugation of a tubulysin analog-linker to Adnectins comprising a Cterminal cysteine was conducted as follows:

A sample of the adnectin to be conjugated to the tubulysin analog wastreated with 5 mM TCEP and incubated at room temperature forapproximately 1 h. TCEP was removed using a G25 gel filtration column(GE Healthcare) equilibrated with 50 mM NaOAc/10 mM NaCl pH 5.5. Thetubulysin analog was dissolved in 100% DMSO and added to a finalconcentration of 5× molar and the reaction was incubated for 2 hours atRT followed by overnight at 4° C. To remove unreacted tubulsyin analog,the reaction mixture was re-applied to the SP cation exchange column asdescribed above.

Adnectins, e.g., GPC3 Adnectins, containing two cysteine residues nearthe C-terminus were conjugated to two molecules of tubulysin analogusing the same methodology described above to generate DAR2(Drug-Adnectin Ratio 2) Adnectins.

Protein concentration was determined using a Nanodrop 8000spectrophotometer (Thermo Scientific). The molecular weight ofconjugated and unconjugated Adnectin was determined by LC-massspectrometry using an Agilent Technologies 6540 UHD Accurate Mass Q-ToFLC-MS equipped with a Zorbax C8 RRHD column.

Using these techniques of expressing, purifying, conjugating andalkylating Adnectins, the Adnectins and Adnectin-drug conjugates listedin Table 3 were prepared.

TABLE 3 Control and Anti-Glypican-3 Binding Adnectins C-term ADX IDelements Modifications Protein sequence ADX_6561_A01 Cys, His₆MGVSDVPRDLEVVAATPTSLLISWSGDYH PHRYYRITYGETGGNSPVQEFTVPGEHETATISGLKPGVDYTITVYAVTYDGEKADKYPP ISINYRTPCHHHHHH* (SEQ ID NO: 94)ADX_6077_F02 Cys, His₆ none MGVSDVPRDLEVVAATPTSLLISWSDDYH ADX_6077_F02Cys, His₆ alkylated AHRYYRITYGETGGNSPVQEFTVPGEHVT ADX_6077_F02 Cys, His₆Drug/linker ATISGLKPGVDYTITVYAVTYDGEKAATD DAR1WSISINYRTPCHHHHHH* (SEQ ID NO: 11) ADX_6077_F02 Cys noneGVSDVPRDLEVVAATPTSLLISWSDDYHA HRYYRITYGETGGNSPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDGEKAATDWSI SINYRTP* (SEQ ID NO: 104) ADX_6912_G022Cys, none MGVSDVPRDLEVVAATPTSLLISWSDDYH His₆AHRYYRITYGETGGNSPVQEFTVPGEHVT ADX_6912_G02 2Cys, alkylated (2x)ATISGLKPGVDYTITVYAVTYDGEKAATD His₆ WSISINYRTPCPPPPPCHHHHHH* (SEQ IDADX_6912_G02 2Cys, Drug/linker (2x) NO: 127) His₆ DAR2 ADX_6912_G02 2CysNone MGVSDVPRDLEVVAATPTSLLISWSDDYH AHRYYRITYGETGGNSPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDGEKAATD WSISINYRTPCPPPPPC* (SEQ ID NO: 126)ADX_6093_A01 Cys, His₆ none MGVSDVPRDLEVVAATPTSLLISWDAPAV ADX_6093_A01Cys, His₆ alkylated TVRYYRITYGETGGNSPVQEFTVPGSKSTA ADX_6093_A01 C-termDrug/linker TISGLKPGVDYTITVYAVTGRGESPASSKPI His₆ DAR1SINYRTPCHHHHHH* (SEQ ID NO: 348) ADX_6093_A01 Cys noneGVSDVPRDLEVVAATPTSLLISWDAPAVT VRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGESPASSKPISI NYRTPC* (SEQ ID NO: 349) 2Cys, noneMGVSDVPRDLEVVAATPTSLLISWDAPAV His₆ TVRYYRITYGETGGNSPVQEFTVPGSKSTA 2Cys,alkylated (2x) TISGLKPGVDYTITVYAVTGRGESPASSKPI His₆SINYRTPCPPPPPCHHHHHH* 2Cys, Drug/linker (2x) (SEQ ID NO: 350) His₆ DAR2

Example 4: In Vitro Characterization of Anti-GPC3 Adnectins andGPC3-AdxDCs

Size Exclusion Chromatography:

Standard size exclusion chromatography (SEC) was performed on candidateAdnectins resulting from the midscale process. SEC of midscaled materialwas performed using a Superdex 200 10/30 or on a Superdex 75 10/30column (GE Healthcare) on an Agilent 1100 or 1200 HPLC system with UVdetection at A214 nm and A280 nm and with fluorescence detection(excitation 280 nm, emission 350 nm). A buffer of 100 mM sodiumsulfate/100 mM sodium phosphate/150 mM sodium chloride, pH 6.8 was usedat the appropriate flow rate for the SEC column employed. Gel filtrationstandards (Bio-Rad Laboratories, Hercules, Calif.) were used formolecular weight calibration.

Thermostability:

Thermal Scanning Fluorescence (TSF) analysis of HTPP Adnectins wasperformed to screen them by relative thermal stability. Samples werenormalized to 0.2 mg/ml in PBS. 1 μl of Sypro orange dye diluted 1:40with PBS was added to 25 μl of each sample and the plate was sealed witha clear 96 well microplate adhesive seal. Samples were scanned using aBioRad RT-PCR machine by ramping the temperature from 25° C.−95° C., ata rate of 2 degrees per minute. The data was analyzed using BioRad CFXmanager 2.0 software. The values obtained by TSF have been shown tocorrelate well with Tm values obtained by DSC over a melting range of40° C. to 70° C. This is considered the acceptable working range forthis technique. A result of ND (“No data”) is obtained when the slope ofthe transition curve is too small to allow its derivative peak (the rateof change in fluorescence with time) to be distinguished from noise. An“ND” result cannot be interpreted as an indication of thermostability.Differential Scanning Calorimetry (DSC) analyses of dialyzed HTPP'd andmidscaled Adnectins were performed to determine their respective Tm's. A0.5 mg/ml solution was scanned in a VP-Capillary Differential Scanningcalorimeter (GE Microcal) by ramping the temperature from 15° C. to 110°C., at a rate of 1 degree per minute under 70 p.s.i pressure. The datawas analyzed vs. a control run of the appropriate buffer using a bestfit using Origin Software (OriginLab Corp).

SPR Affinity Measurements:

Surface plasmon resonance (SPR) was performed to calculate off-rates(k_(d)) and binding affinities of α-GPC3 adnectins andtubulysin-conjugated AdxDCs using a Biacore T100 instrument (GEHealthcare). Recombinant human (aa 1-559) and murine (aa 25-557)glypican-3 proteins (R&D Systems) were diluted to 10 g/ml in 10 mMsodium acetate pH 4.5 and individually immobilized onto active flowcells of a CM5 biosensor following the manufacturer's amine couplingprotocol (GE Healthcare), targeting -1000RU immobilization density ofeach protein per flow cell. SPR experiments were conducted at 37° C.using HBS-P+(10 mM HEPES, 150 mM NaCl, 0.05% (v/v) Surfactant P20, pH7.4) running buffer (GE Healthcare). For affinity measurements, aconcentration series of 200-1.56 nM α-GPC3 adnectins and AdxDCs wereprepared in running buffer and injected at 30 l/min across the human andmurine GPC3 biosensor flow cells. For off-rate measurements, single 200nM adnectin/AdxDC concentrations were injected using identicalconditions. One 30s injection of 10 mM glycine pH 1.7 was used to removebound adnectin and regenerate the GPC₃ surfaces between assay cycles.

Rate constants k_(a) (k_(on)) and k_(d) (k_(off)) were derived fromreference-subtracted sensorgrams fit to a 1:1 binding model in BiacoreT100 Evaluation Software v2.0.4 (GE Healthcare). The affinity constant,K_(D) was calculated from the ratio of rate constants k_(d)/k_(a).

Cell Binding Assay:

The binding of GPC3 adnectins to huGPC3 positive cells Huh7 wasevaluated by flow cytometry essentially as follows. Huh7 carcinoma cellsgrown in DMEM media with 10% FBS. Cells were harvested using Versene, anEDTA cell dissociation solution from Lonza, Cat. #17-711E. Tumor cells(1E5cells/reaction) were suspended in FACS buffer (PBS, 1% BSA, 0.05% NaAzide) and mixed with a serial dilution of AdxDC for one hour on ice.Cells were washed three times with FACS buffer, and bound AdxDC wasdetected with an in house anti-scaffold monoclonal Ab and PE-conjugatedAntibody from (RnD Systems), cat # NL007, and read on a flow cytometer.Data analysis was done using FlowJo Software, and EC50 of 50% of maximumbinding was determined using PRISM™ software, version 5.0 (GraphPadSoftware, La Jolla, Calif., USA).

Cell Growth Inhibition Assay:

A ³H thymidine assay, where the inhibition of incorporation of ³Hthymidine indicates inhibition of proliferation of the tested cell line,was used to assess the dose-dependent inhibitory effect of the AdxDC onthe proliferation of Hep3B, Huh7and HepG2 cells. The human tumor celllines were obtained from the American Type Culture Collection (ATCC),P.O. Box 1549, Manassas, Va. 20108, USA, and cultured according toinstructions from ATCC. Cells were seeded at 1.25×10⁴ cells/well in96-well plates, and 1:3 serial dilutions of GPC3 AdxDC were added to thewells. Plates were allowed to incubate for 72 h. The plates were pulsedwith 1.0 μCi of ³H-thymidine per well for the last 24 hours of the totalincubation period, harvested, and read on a Top Count ScintillationCounter (Packard Instruments, Meriden, Conn.). The EC₅₀ values—theAdnectin drug conjugate concentration at which 50% of maximum cellproliferation inhibition was achieved—were determined using PRISM™software, version 4.0 (GraphPad Software, La Jolla, Calif., USA).

A summary of the in vitro properties of DAR1 and DAR2 forms of the AdxDCconjugate is summarized in Table 4.

TABLE 4 In vitro characterization of GPC3-Tubulysin AdxDC Non-bindingcontrol Glypican-3-binding Adnectin ID ADX_6093_A01 ADX_6077_F02Conjugate DAR1 DAR1 DAR2 DAR2 % monomer 100% 100% DSC T_(m) (PBS) 86° C.89° C., 93° C. SPR K_(D) (37° C.) No binding 12 nM (hu)  11 nM (mu) SPRk_(off) (37° C.) No binding 9.8 × 10⁻⁴ s⁻¹ (hu)  7.7 × 10⁻⁴ s⁻¹ (mu)Adnectin No binding 5 nM (hu; Huh7)^(alk) cell-binding EC₅₀ up to ~200nM^(alk) AdxDC No effect 0.2 nM (hu; Huh7, HepG2) cell-killing IC₅₀ upto ~25 nM  ^(alk)measured for alkylated (capped Adnectin); all othermeasurements for DAR1 AdxDC (no PEG)

Example 5: Cell Binding of GPC3-AdxDC to Human Hep3B and H446 TumorCells

GPC3 AdxDC were evaluated by flow cytometry for binding to human Hep3Bhepatocellular carcinoma cells grown in MEM with 10% FBS, and H446 smallcell lung carcinoma cells grown in RPMI with 10% FBS. Cells wereharvested using Cellstripper, a non-enzymatic cell dissociation solutionfrom Mediatch (Corning: Manassas, Va. 20109), Cat. #25-056-CL. Tumorcells (25,000/reaction) were suspended in FACS buffer (PBS+5% FBS+0.01%NaN₃) and mixed with a serial dilution of AdxDC for 1 hour on ice. Cellswere washed three times with FACS buffer, and bound AdxDC was detectedwith His Tag PE-conjugated Antibody from R&D System, cat # IC050P, andread on a flow cytometer. Data analysis was done using FlowJo Software,and EC50 of 50% of maximum binding was determined using PRISM™ software,version 5.0 (GraphPad Software, La Jolla, Calif., USA).

The results, which are shown in FIG. 4A-D, show that ADX_6077_F02 AdxDCDAR1 and DAR2 bind to both types of human tumor cells.

Example 6: GPC3 AdxDC Inhibit Cell Growth of Hep3B, H446 and HepG2 TumorCells

This Example shows that GPC3 AdxDC DAR1 and DAR2 inhibit cellproliferation of Hep3B (Glypican3 high) HCC cells, H446 (Glypican3 low)SCLC Cells and HepG2 tumor cells. Thymidine incorporation assays wereconducted as described above. The results, which are shown in FIGS. 5A-Band 6A-B show that GPC3 AdxDC DAR1 and DAR2 inhibit cell growth of thethree different cell lines, but that the control AdxDC adnectinconjugate does not inhibit growth of these cells.

Example 7: Cell Surface Binding Assay Time Course for GPC3-AdxDC

To ensure maximum target engagement prior to internalization studies,binding of ADX_6077_F02 DAR1 (i.e., with a “PC” terminus, but notconjugated) to GPC-3 positive cells Hep3B was determined using thefollowing binding assay: AF-488 fluorescently labeled adnectinADX_6077_F02 and negative control (NBC) ADX_6093_A01 were used in Hep3Bcell binding assay. For binding analysis, Hep3B cells were plated into a384 well plate, incubated for 16h to allow cells to adhere and then thecells were fixed with 2% formaldehyde. ADX_6077_F02 and ADX_6093_A01 at100 nM were added into the cell plate and incubated at room temperaturefor 7 time points: 0 minute, 10 minutes, 15 minutes, 20 minutes, 60minutes, 120 minutes and 180 minutes. After binding, the cells werewashed with phosphate-buffered saline (PBS) twice and total cellfluorescence intensity per cell was then measured using high contentanalysis.

The results, indicate that ADX_6077_F02 demonstrated fast associationonto cell surfaces. Two hours was found to be sufficient to reach agreater than 95% binding plateau using 100 nM of the Adnectin.

Example 8: Kinetic Internalization of GPC3-AdxDC

To quantify anti-glypican 3 adnectin induced internalization, ahigh-content Alexa quenching assay was applied. Hep3B and H446 cellswere seeded in 384 well plates and incubated for 16 hours at 37° C.AF-488 fluorescently labeled ADX_6077_F02 DAR1 at 100 nM were then addedinto the cell plates and incubated at 37° C. for the indicated timeprior to fixation and quenching. Internalized Adnectin was measured asincreased fluorescence above the unquenchable signal. Total fluorescencefrom “unquenched control” at each time point was monitored in parallelto be used as indicator of the amount of adnectin initially bound to thecells. The images of the cells were taken by Arrayscan to show thelocalization of the adnectin, and used for cell fluorescence intensityquantification.

Quantification studies confirmed high expression levels of the GPC3receptor on Hep3B (approximately 1.1×10⁶ active binding copies/cell) andlower levels on H446 cells (approximately 2.6×10⁵ active bindingcopies/cell). Following fixation, total and intracellular FL weredetermined and used to measure internalization of the Adnectinmolecules.

The results of these assays indicate that the anti-GPC3 adnectin isinternalized by Hep3B and H446 cells (FIG. 7) at a medium-slow rate(T_(1/2)>1 hr) and reaches >90% internalization after 6 hours. As shownin FIG. 8, at the 15 minute time point, most of the anti-GPC3 adnectinis membrane associated, and by the 8 hour time point most of theGPC3-Adectin signal is inside of the cells.

Example 9: In Vivo Pharmacokinetics of GPC3-AdxDC

The systemic exposure profile of anti-GPC3 AdxDC (DAR1) was determinedin mice. Female NOD/SCID mice (13 weeks of age) were dosed intravenouslywith a single dose of high (240 nmol/kg) and low (24 nmol/kg) doses ofGPC3-binding and non-binding-control AdxDCs (GPC3 DAR1 AdxDC and RGEAdxDC, respectively) as per the experimental design below. The indicatedblood time points were serial tail vein collections using CPDanticoagulant (Citrate-phosphate-dextrose solution, Sigma C7165). Plasmaobtained from these blood samples were aliquoted and stored at -80 Cuntil ready for analysis.

TABLE 5 Dosing Schedule for Xenograft Model Group N Test Article Dose(nmol/kg) 1 Low dose 3 Glypican3 binding AdxDC 24 1 High Dose 3 240Timepoints: 5 - 20-40 min, 1 - 1.5 - 2 - 3 - 4 - 6 - 8-24 h

AdxDC plasma levels where analyzed using Mesoscale (MSD) ligand bindingassays with two different formats. MSD assays for total levels ofconjugated and unconjugated Adnectin assays used for capture an in housegenerated anti-His monoclonal antibody (at 4 ug/ml), and for detection apooled in-house generated rabbit anti-scaffold polyclonal at 1:10000dilution followed by a goat anti-rabbit sulfotagged antibody (at 1ug/ml). MSD assays for intact conjugated Adnectin used for capture anin-house generated anti-His monoclonal antibody (at 4 ug/ml), and fordetection an in-house generated sulfotagged mouse anti-tubulysinantibody (at 1 ug/ml).

The results of this assay, which are summarized in Table 6 (Noncompartmental Phoenix WinNonlin analysis, NCA model) and FIG. 9(Anti-tubulysin MSD assay), further indicate that the AdxDC has a shortexposure profile in mice.

TABLE 6 Pharmacokinetics Parameter Summary of GPC3 AdxDC NCA AUC_%HL_Lambda_z Cl_obs Vss_obs AUCall AUCINF_obs Extrap_obs MRTINF_obs Cmaxdose species (h) (mL/h/kg) (mL/kg) (h*nmol/L) (h*nmol/L) (%) (h)(nmol/L) high total 0.74 288 136 835 836 0.11 0.47 2124 intact 0.80 353249 740 740 0.06 0.71 1870 low total 0.59 344 192 69 70 1.12 0.56 165intact 0.63 445 233 58 59 0.48 0.52 145

Example 10: Inhibition of Tumor Growth in Rodent Xenograft Models

The efficacy of unPEGylated GPC3-tubulysin drug conjugate was tested inCD1 mice and Fischer rats.

NOD-SCID and CD1 female mice (13 weeks old, from Charles RiverLaboratories, Wilmington, Mass.) and female Fischer rats (10 weeks ofage, from Charles River laboratories, Wilmington, Mass.) were housed ina temperature-controlled room with a reversed 12 hour light/dark cycle.Water and standard chow food were available ad libitum. Animals forsafety studies were randomized and distributed between treatment groupsto receive either control or test AdxDC based on body weight (about20-25 g).

Hep3B, a human hepatocellular carcinoma, was maintained in culture usingEMEM Cat # ATCC 30-2003 supplemented with 10% FBS (Thermo Cat #ATK-33398). For efficacy studies, xenografts were generated bysubcutaneous implantation of 100 ul of Hep3B 5×10⁶ cells (50% cellularsuspension with Standard Phenol Red Matrigel, Corning Cat #354234) inthe right flank of NOD-SCID mice. In order to demonstrate in vivoefficacy, the AdxDCs were administered by intravenous injection ineither 50 mM NaOAc/150 mM NaCl/pH 5.5, or Phosphate-Buffered Saline(PBS). Controls were treated with a non-binding control AdxDC. Testanimals (n=8 animals/group) were dosed intravenously every three dayswith a total of six doses, with varying dosages of the AdxDC. Bodyweight measurements were recorded pre-randomization, on randomizationday, and two times a week during the treatment periods and at the end ofthe study. Tumor growth was monitored using digital caliper measurementstwice a week. The results were assessed using student's t-test 2 tailedpaired analysis. Representative study design and results using everythree days dosing administration are represented in Table 7 and FIG. 10.

TABLE 7 Dosing Schedule Dose T/C (d 17) AdxDC DAR μmol/kg Schedule (%)ADX_6093_A01-961 1 0.3 Every 3 days (non-binding control)ADX_6077_F02-961 1 0.1 Every 3 days 96 ADX_6077_F02-961 1 0.3 Every 3days 95 ADX_6912_G02-961 2 0.3 Every 3 days 96 ADX_6912_G02-961 2 0.3Every 5 days 96

Weekly administration was evaluated in Hep3B xenografts. The resultsindicate that QW administration of ADX_6077_F02-961 DAR1 and DAR2 at 0.1μmol/kg effectively inhibited HepG2 xenografts TV₀=380-480³ (FIG. 11),TV₀=228-350 mm³ (FIG. 12), and TV₀=514-673 mm³ (FIG. 13).

In summary, weekly administration of GPC3 AdxDCs inhibits growth of HCCtumor xenografts, and DAR1 and DAR2 GPC3 AdxDCs demonstrated equivalenttumor growth inhibition, which was target-dependent. In addition, theprevention of tumor burden-induced weight loss was associated with theanti-tumor activity of the GPC3 AdxDCs.

In mice safety studies, CD1 mice treated intravenously every other dayfor a total of 9 doses at varying doses up to a highest dose of0.5umol/kg, 5× the efficacious dose (0.1 umol/kg) with both GPC3 andnon-binding control AdxDCs. No MTD was identified in the CD1 mice safetystudies. No kidney toxicity was observed for any group, at any dose orfrequency. In CD1 mice, the half-life of the AdxDC was approximately 20minutes (MSD assays as described above). No body weight loss wasobserved and all mice survived treatment to scheduled necroscopy. Serumchemistry and hematology were evaluated at intervals through the dosingperiod using Abaxis Veterinary Diagnostics instruments, VETSCAN VS2 andHM5, respectively. There were no significant differences observed inserum chemistry or hematology compared to baseline. Histopathology wasevaluated via H&E staining of heart, liver, spleen and kidney tissuescollected at the end of the study. No dose-limiting toxicities wereobserved in any of the evaluated tissues, and minimal/mild tubularepithelium neuropathy was observed in all groups.

In Fischer rat safety studies, the half life of the AdxDC wasapproximately 30 minutes (MSD assays as described above). Some dosedependent tolerability and skeletal muscle degeneration were observed atthe most frequent administration (every other day) of doses of0.36umol/kg with no changes in bone marrow or liver histopathology orheart toxicity. These findings were not observed when the same ratstudies were conducted using weekly administration of AdxDCs at the samedosage range.

Overall, excellent efficacy despite the short plasma half-life and lowoff-target toxicity, consistent with low systemic exposure, was observedin both rodent species.

Example 11: Mapping of Adnectin Binding Site on Human GPC3 Using HDX-MS

The Adnectin binding site on human GPC3 (amino acid sequence shown inFIG. 14) was evaluated using hydrogen-deuterium exchange massspectrometry (HDX-MS). The hydrogen/deuterium exchange mass spectrometry(HDX-MS) method probes protein conformation and conformational dynamicsin solution by monitoring the deuterium exchange rate and extent in thebackbone amide hydrogens. The level of HDX depends on the solventaccessibility of backbone amide hydrogens and the conformation of theprotein. The mass increase of the protein upon HDX can be preciselymeasured by MS. When this technique is paired with enzymatic digestion,structural features at the peptide level can be obtained, enablingdifferentiation of surface exposed peptides from those folded inside, orfrom those sequestered at the interface of a protein-protein complex.Typically, the deuterium labeling and subsequent quenching experimentsare performed, followed by online pepsin digestion, peptide separation,and MS analysis.

Prior to mapping the Adnectin binding site on human GPC3 recognized byADX_6077_F02 by HDX-MS, non-deuteriated experiments were performed togenerate a list of common peptic peptides for GPC3 samples, achieving asequence coverage of 87.4% for GPC3 (FIG. 14). In this experiment, 10 mMphosphate buffer (pH 7.0) was used during the labeling step, followed byadding quenching buffer (200 mM phosphate buffer with 4M GdnCl and 0.4MTCEP, pH 2.5, 1:1, v/v).

For Adnectin binding site mapping experiments, 5 μL of each sample (GPC3or GPC3 with ADX_6077_F02) was mixed with 55 μL HDX labeling buffer (10mM phosphate buffer in D2O, pD 7.0) to start the labeling reactions. Thereactions were carried out for different periods of time: 1 min, 10 min,and 240 min. By the end of each labeling reaction period, the reactionwas quenched by adding quenching buffer (1:1, v/v) and the quenchedsample was injected into Waters HDX-MS system for analysis. The observedcommon peptic peptides were monitored for their deuterium uptake levelsin the absence/presence of ADX_6077_F02 (FIGS. 14 and 15).

Experimental data obtained from HDX-MS measurements indicate thatAADX_6077_F02 recognizes a discontinuous Adnectin binding site comprisedof two peptide regions in human GPC3:

-   -   Region 1: HQVRSFF (amino acid residues 36-42 of GPC3); SEQ ID        NO: 356    -   Region 2: EQLLQSASM (amino acid residues 90-98 of GPC3); SEQ ID        NO: 346

Example 12: Generation of DG Variants

Analysis of the amino acid sequence of ADX_6077_F02 indicated that theDG in the FG loop of the molecule might be at low risk of aspartateisomerization.

(SEQ ID NO: 118) MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDGEKAATDWSISINYRTPCHH HHHHEight variants of ADX_6077_F02 with mutations at the DG site weregenerated. The sequences of these mutants are summarized in Table 8.

TABLE 8 ADX_6077_F02 Variants C-term elements ModificationProtein sequence DG→EG Cys alkylated GVSDVPRDLEVVAATPTSLLISWSDDYHAHmutant RYYRITYGETGGNSPVQEFTVPGEHVTATIS GLKPGVDYTITVYAVTYEGEKAATDWSISINYRTPC* (SEQ ID NO: 143) DG→SG Cys alkylatedGVSDVPRDLEVVAATPTSLLISWSDDYHAH mutant RYYRITYGETGGNSPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYSGEKAATDWSISIN YRTPC* (SEQ ID NO: 169) DG→AG Cysalkylated GVSDVPRDLEVVAATPTSLLISWSDDYHAH mutantRYYRITYGETGGNSPVQEFTVPGEHVTATIS GLKPGVDYTITVYAVTYAGEKAATDWSISINYRTPC* (SEQ ID NO: 197) DG→GG Cys alkylatedGVSDVPRDLEVVAATPTSLLISWSDDYHAH mutant RYYRITYGETGGNSPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYGGEKAATDWSISI NYRTPC* (SEQ ID NO: 224) DG→DS Cysalkylated GVSDVPRDLEVVAATPTSLLISWSDDYHAH mutantRYYRITYGETGGNSPVQEFTVPGEHVTATIS GLKPGVDYTITVYAVTYDSEKAATDWSISINYRTPC* (SEQ ID NO: 251) DG→DA Cys alkylatedGVSDVPRDLEVVAATPTSLLISWSDDYHAH mutant RYYRITYGETGGNSPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDAEKAATDWSISIN YRTPC* (SEQ ID NO: 278) DG→DL Cysalkylated GVSDVPRDLEVVAATPTSLLISWSDDYHAH mutantRYYRITYGETGGNSPVQEFTVPGEHVTATIS GLKPGVDYTITVYAVTYDLEKAATDWSISINYRTPC* (SEQ ID NO: 305) DG→DV Cys alkylatedGVSDVPRDLEVVAATPTSLLISWSDDYHAH mutant RYYRITYGETGGNSPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDVEKAATDWSISIN YRTPC* (SEQ ID NO: 332)

Example 13: Biophysical Characterization of DG Variants

One-hundred to one-hundred-fifty milligrams of each of the eight mutantswas made, purified and alkylated as previously above. Three to fivemilligrams of each of eight alkylated variants at 1-3 mg/mL weresubjected to SEC, DSC, GPC3 binding (SPR 1pt off-rates), MS and HIC. Theresults are summarized in Table 9.

TABLE 9 Biophysical properties of ADX_6077_F02_DG Variants huGPC3 muGPC3mono k_(off) k_(off) % T_(m) Mutant Clone ID (mut/par) (mut/par) (SEC)(DSC) DG→EG P1-055673 5.4 5.1 96% 88° C. DG→SG P1-055668 5.2 4.8 96% 85,91° C. DG→AG P1-055669 5.9 5.5 96% 85, 91° C. DG→GG P1-055670 14 12 96%84, 90° C. DG→DS P1-055667 3.5 3.3 96% 84° C. DG→DA P1-055660 3.9 3.796% 86, 98° C. DG→DL P1-055671 3.6 3.1 96% 84° C. DG→DV P1-055672 7.97.0 96% 84° C.

Six of the eight DG mutants demonstrated a 3-5 fold increase in k_(off)compared to the parental adnectin, and were monomeric and thermostable.The binding affinities of the alkylated GPC3 DG mutant adnectins forhuman and murine GPC3 were further evaluated by Biacore T100 usingHBS-P+ running buffer with direct immobilization of human and mouse GPC3proteins [Hu (Fc 2,3) and Mu (Fc 4) GPC3-His (R&D Systems)] with a200-1.56 nM series injected for 180s association, 600s dissociation. Thedata fit a 1:1 binding model in BiaEvaluation software and is summarizedin Table 10.

TABLE 10 Binding Kinetics of DG Mutant Adnectins huGPC3 muGPC3 Foldaffinity Fold affinity (KD) (KD) difference vs difference vs alkylatedalkylated Description ka (1/Ms) kd (1/s) KD (M) parent ka (1/Ms) kd(1/s) KD (M) parent ADX_6077_F02 9.53E+04 5.34E−04 5.60E−09 1.0 1.3E+054.9E−04 3.8E−09 1.0 alkylated 6077_F02 7.22E+04 1.83E−03 2.54E−08 4.51.0E+05 1.7E−03 1.6E−08 4.3 DG−>DA 6077_F02 7.16E+04 1.66E−03 2.33E−084.2 1.0E+05 1.5E−03 1.5E−08 3.9 DG−>DS 6077_F02 9.40E+04 2.21E−032.35E−08 4.2 1.3E+05 2.0E−03 1.6E−08 4.1 DG−>SG 6077_F02 1.03E+052.43E−03 2.37E−08 4.2 1.4E+05 2.1E−03 1.5E−08 4.0 DG−>AG 6077_F027.26E+04 1.59E−03 2.19E−08 3.9 1.0E+05 1.5E−03 1.5E−08 3.8 DG−>DL6077_F02 7.10E+04 3.09E−03 4.35E−08 7.8 8.5E+04 2.5E−03 3.0E−08 7.8DG−>DV 6077_F02 7.63E+04 2.29E−03 3.00E−08 5.3 1.1E+05 2.1E−03 1.9E−085.1 DG−>EG

The data demonstrated that these GPC3 DG mutants have approximately 3-5fold decreased affinity for human and murine GPC3 compared to parentalADX_6077_F02. The differences in affinities were driven by fasteroff-rates, whereas the on-rates were consistent with the parentaladnectin (FIG. 16).

Example 14: Cell Binding and Immunogenicity Assessment of DG Variants

The binding of DG variants to huGPC3 in DG GPC3 AdxDCs mutants wereevaluated by flow cytometry for binding to Huh7 carcinoma cells grown inDMEM media with 10% FBS. Cells were harvested using Versene, an EDTAcell dissociation solution from Lonza, Cat. #17-711E. Tumor cells(1EScells/reaction) were suspended in FACS buffer (PBS, 1% BSA, 0.05% NaAzide) and mixed with a serial dilution of AdxDC for one hour on ice.Cells were washed three times with FACS buffer, and bound AdxDC wasdetected with an in house anti-scaffold monoclonal Ab and PE-conjugatedAntibody from (RnD Systems), cat # NL007, and read on a flow cytometer.Data analysis was done using FlowJo Software, and EC50 of 50% of maximumbinding was determined using PRISM™ software, version 5.0 (GraphPadSoftware, La Jolla, Calif., USA).

For anti-His detection of (non DG mutants), the same protocol was used,except in house generated APC-conjugated anti-His antibody was used.

The results, which are shown in Table 11, indicate that the mutants hadsimilar EC50 values as that of the parent Adnectin.

The DG variants to huGPC3 were also assessed for their potential toelicit an immune response in humans using a human PBMC proliferationassay. PBMCs from 40 donor with HLA Class II haplotypes closely matchingthe world population frequencies were cultured in the presence of the DGvariants or controls for 7 days. At the end of the assay, CFSE-labeledCD4+T cells were analyzed by FACS for proliferation. The percentage ofdonors that showed proliferating CD4+T cells were analyzed as a read-outfor human immunogenicity risk. Assay results indicate that the DG to DAmutant (PI-055660) has a significantly lower risk for immunogenicity(IMG) (18% of donors responded positive) compared to the other DGmutants (36-54% positive responses) as summarized in Table 11.

TABLE 11 Cell Binding Kinetics of DG Variants huGPC3 on cells Clone IDEC₅₀ IMG: % +ve Mutant P1- (mutant/parent) responders DG→EG 055673 1.649% DG→SG 055668 1.1 41% DG→AG 055669 1.1 54% DG→DS 055667 1.4 36% DG→DA055660 1.3 18% DG→DL 055671 1.7 54%A summary of the characteristics of the DA variant AdxDC DAR1(“GPC3_AdxDC DA variant-DAR1” or “DA variant AdxDC DAR1”) are set forthin Table 12:

TABLE 12 Characteristics of the DA variant AdxDC DAR1 % monomer 100% DSCT_(m) (PBS) 79, 87° C. SPR K_(d) 25 nM (hu, 37° C.)  23 nM (mu, 37° C.)SPR k_(off) 2.1 × 10⁻³ s⁻¹ (hu, 37° C.)  9.5 × 10⁻⁴ s⁻¹ (mu, 37° C.)Cell-binding EC₅₀ (Huh7) 2.2 nM * Cell-killing IC₅₀ (Huh7) 0.2 nM   *Measured for unconjugated protein

Example 15: FITC Labeled and PEGylated Anti-GPC3 Adnectins

FITC Labeling.: ADX_6077_F02 and non-binding control Adnectin werereduced with DTT or TCEP followed by G25 gel filtration chromatographyor dialysis. Excess Fluorescein-5-maleimide reagent (Thermo Scientific)was then added and the mixture incubated at room temperature forapproximately 2 hours followed by G25 gel filtration chromatography orextensive dialysis (3-4 buffer changes). The resulting degree oflabeling was measured by absorbance following the manufacturer'sinstructions and/or by mass spectrometry. The binding affinities ofFITC-labeled and PEGylated GPC3 for human and murine GPC3 was evaluatedas described in the previous Examples, and the results are summarized inTable 13.

TABLE 13 Kinetics of Modified GPC3 Adnectins huGPC3 muGPC3 Adnectin ka(1/Ms) kd (1/s) KD (M) ka (1/Ms) kd (1/s) KD (M) ADX_6077_F02 8.90E+045.24E−04 5.88E−09 1.23E+05 4.53E−04 3.69E−09 alkylated 6077_F02-FITC8.15E+04 4.98E−04 6.11E−09 1.12E+05 4.46E−04 3.97E−09 RGE-FITC Nobinding No binding 6077_F02.dPEG 7.35E+04 6.02E−04 8.19E−09 1.03E+055.59E−04 5.43E−09 6077_F02.dPEG 7.50E+04 6.07E−04 8.10E−09 1.06E+055.57E−04 5.26E−09 6077_F02-PEG3.4 7.18E+04 5.84E−04 8.14E−09 1.02E+055.23E−04 5.12E−09

The data demonstrated that both FITC-labeled and PEGylated anti-GPC3adnectins retained binding affinity to both human and murine GPC3.

Example 16: Additional Characteristics of GPC3_AdxDC DA and DG Molecules

The GPC3_AdxDC DA variant-DAR1 was shown to be chemically andbiophysically stable at pH 6.0, in accelerated-stability studies. Inaddition, its affinity for human GPC3 (by SPR) was unchanged after 4weeks at 40° C.

Aspartate isomerization of the DA variant was about 4 fold lower thanthat of the parent DG molecule. The percent isomerization of D80 of theDG molecule, after incubation for 3 weeks at 40° C. at pH 6 or pH 7 was3.6 and 2.4, respectively.

The GPC3_AdxDC (DG) shows a favorable toxicity profile under weeklydosing in CDF rats (Q7Dx4) (Table 14). No adverse responses were seen inhematology or serum chemistry profiles in CDF rats under weeklyadministration of GPC3 AdxDC (Q7Dx4).

TABLE 14 Toxicity profile of GPC3_AdxDC (DG) Skeletal Heart MuscleTreatment (degener- Liver (regener- Kidney (umol/kg) ation) (↑ mitosis)ation) (↑ mitosis) NBC* 0.28 minimal none none minimal AdxDC GPC3 0.093none none none none AdxDC 0.28 none none none minimal *“NBC” refers tonon-binding AdxDC (Adnectin Drug Conjugate)

Example 17: GPC3 Adnectin Drug Conjugates Bind Human GPC3 XenograftTissue In Vivo

Human GPC3 high expression Hep3B xenograft tissue was incubated withFITC-conjugated GPC3-binding Adnectin DG molecule (“GPC3_AdxDC (DG)”)DAR1 at a concentration of 0.04 μg/ml or with a non GPC3 bindingAdnectin at 0.2 μg/ml. The results indicate that the GPC3_AdxDC (DG)molecule binds Hep3B xenograt tissue, whereas the non-binding Adnectindid not significantly bind.

Other tissues were also tested for binding, and the results indicatethat there is some non-specific binding of GPC3_AdxDC (DG) to placenta.However, the molecule does not bind significantly to stomach, heart,kidney, liver, skin or tonsil tissue.

GPC3_AdxDC (DA) DAR1 shows similar but weaker binding in XenograftHep3B. Saturated binding was achieved at 0.2 μg/ml.

Example 18: GPC3_AdxDC (DA) is Highly Efficacious in Cell-Line-DerivedXenografts with High Expression of Glypican-3

Hep3B (hepatocellular carcinoma; 260,000 GPC3 molecules/cell) xenograftswere used in NSG mice. GPC3_AdxDC (DA) DAR1 or a non-binding controlAdnectin were administered i.v. weekly, 3 times, at the doses indicatedin Table 15.

TABLE 15 Dosage and tumor growth inhibition of Hep3B xenografts in NSGmice Dose TGI_(D 26)* AdxDC mpk μmol/kg (%) GPC3_AdxDC (DA) 1.4 0.12 1090.5 0.04 103 0.1 0.01 62 0.05 0.004 20 Non-binding Adnectin 1.4 0.12 —(RGE; −ve control) TGI_(D26)*: Tumor Growth Inhibition at Day 26

The results, which are shown in Table 15 and FIG. 17, indicate thatGPC3_AdxDC (DA) is effective in inhibiting Hep3B tumor growth in vivo.

A similar experiment was conducted with cell-line-derived xenograftswith low expression of Glypican-3 (H446). H446 cells are small-cell lungcarcinoma cells with about 40,000 human PC3 molecules/cell. The cellswere injected into CB17 SCID mice.

TABLE 16 Dosage and tumor growth inhibition of H446 cells in CB17 SCIDmice Dose (Q3Dx4) TGI_(D 21) AdxDC mpk μmol/kg (%) GPC3-binding 12 1.0(BMT-279771) 8 0.67 53 4 0.33 2 0.17 16 RGE 1.4 0.12 — (−ve control)

The results, which are shown in Table 16 and FIG. 18, indicate thatGPC3_AdxDC (DA) slows down the growth of these tumors.

Example 19: Preferential Uptake of GPC3_AdxDC to Hep3B Tumor Relative toNormal Tissues

Mice were dosed with ³H labeled GPC3_AdxDC at 0.015 or 0.22 μmol/kg, andradioactivity was measured by Whole-Body Autoradiography (QWBA) after0.17 hours, 1 hour, 5 hours and 168 hours.

The results, which are shown in FIGS. 19 and 20, indicate the following:

-   -   Rapid distribution to tumor and highly perfused tissues;    -   High level radioactivity remained in the tumors 168 hours after        dosing, with no, or low, radioactivity in other tissues;    -   Significant radioactivity in kidney: about 30% of the        radioactivity was excreted in the urine; and    -   Similar patters of expression between high and low dose groups.

In a similar experiment, mice were dosed with ³H labeled GPC3_AdxDC ornon-binding AdxDC control at 0.22 μmol/kg, and radioactivity wasmeasured by QWBA after 0.17 hours, 1 hour, 5 hours and 168 hours.

The results, which are shown in FIGS. 21 and 22, indicate that there isa higher uptake to Hep3B Tumor with GPC3_AdxDC relative to thenon-binding control (RGE AdxDC). The distribution profile in othertissues is comparable for the GPC3_AdxDC and the non-binding AdxDCcontrol.

The total radioactivity concentration of GPC3_AdxDC in tumor and tissuesis represented in FIG. 23. The figure shows the presence of much higherlevel of GPC3_AdxDC in the tumors than in other tissues (except thekidney).

Example 20: Positional Scanning of Anti-GPC3 Adnectins

This Example describes positional scanning of 6077_F02 in which EIDKPSQ(SEQ ID NO: 369) was removed and PC was added, and wherein amino acid 79(i.e., the “D” of “DG”) is either G (as in original clone) or A.

The two proteins, different only at amino acid 79, were mixed during thelibrary construction. Binding to human glypican-3-biotin was determinedat 100 nM, 10 nM and 1 nM. For each batch, the 10 nM selection elutionwas compared to the flag elution and the 1 nM selection elution was alsocompared to the flag elution. This generated 4 heat maps for each loop:10 nM when 79 is G; 1 nM when 79 is G; 10 nM when 79 is A and 1 nM when79 is A.

For the FG loop, the three segments were combined together to show thefull heat map. For position 79, on heat map was generated where it wasnormalized to the G, and one heat map where it was normalized to the A.

The results, in the form of heat maps, are shown in FIGS. 24-31. In theheat maps, a number >1 indicates a favorable substitution, however, anynumber >0.2 is also acceptable as a substitution The higher the number,the more favorable the substitution. For example, the heat maps indicatethe following for the DG parent adnectin:

-   -   In loop BC (i.e., sequence SDDYHAH (amino acids 15-21 of SEQ ID        NO: 98)):        -   S23 may be substituted with any amino acid;        -   D24, is preferably not substituted with any other amino            acid, although S and E may be acceptable.        -   Other acceptable substitutions may be derived from the heat            maps, wherein any substitution having a number >0.2 is            acceptable and a number >1 is preferable.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

TABLE 2 SUMMARY OF SEQUENCES SEQ ID Description SEQUENCE 1Full length wild-type human ¹⁰Fn3VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPV domainQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISI NYRT 2Core wild-type human ¹⁰Fn3 EVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSdomain KSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT 3Human ¹⁰Fn3 domain six loopVSDVPRDLEVVAA(X)_(u)LLISW(X)_(v)YYRITY(X)_(w)FTV(X)_(x)ATIsequences generically defined SG(X)_(y)YTITVYAV(X_(z)ISINYRT 4Wild-type human ¹⁰Fn3 domain C-VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPV terminal flexible linkerQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISI NYRTEIDKPSQ 5 ADX_4578_F03EVVAATPTSLLISWHPPHPNIVSYHIYYGETGGNSPVQEFTVEGS CoreKSTAKISGLKPGVDYTITVYAVAPEIEKYYQIWINYRT 6 ADX_4578_F03 HPPHPNIVS BC loop7 ADX_4578_F03 EGSKST DE loop 8 ADX_4578_F03 VAPEIEKYYQ DE loop 9ADX_45708_F03 VSDVPRDLEVVAATPTSLLISWHPPHPNIVSYHIYYGETGGNSPV full-lengthQEFTVEGSKSTAKISGLKPGVDYTITVYAVAPEIEKYYQIWINYR T 10 ADX_4578_F03MGVSDVPRDLEVVAATPTSLLISWHPPHPNIVSYHIYYGETGGNSfull-length w/N-terminal leaderPVQEFTVEGSKSTAKISGLKPGVDYTITVYAVAPEIEKYYQIWIN and C-terminal tailYRTEGSGS 11 ADX_4578_F03 VSDVPRDLEVVAATPTSLLISWHPPHPNIVSYHIYYGETGGNSPVfull-length with C-terminal P_(m)X_(n)QEFTVEGSKSTAKISGLKPGVDYTITVYAVAPEIEKYYQIWINYR TP_(m)X_(n) 12ADX_4578_F03 VSDVPRDLEVVAATPTSLLISWHPPHPNIVSYHIYYGETGGNSPVfull-length with C-terminal P_(m)CX_(n)QEFTVEGSKSTAKISGLKPGVDYTITVYAVAPEIEKYYQIWINYR TP_(m)CX_(n) 13ADX_4578_F03 VSDVPRDLEVVAATPTSLLISWHPPHPNIVSYHIYYGETGGNSPVfull-length with C-terminalQEFTVEGSKSTAKISGLKPGVDYTITVYAVAPEIEKYYQIWINYR PmCXn (m = 1; n = 0) TPC14 ADX_4578_F03 VSDVPRDLEVVAATPTSLLISWHPPHPNIVSYHIYYGETGGNSPVfull-length with C-terminalQEFTVEGSKSTAKISGLKPGVDYTITVYAVAPEIEKYYQIWINYRPmCXn (m = 1; n = 0) and His₆ tag TPCHHHHHH 15 ADX_4578_F03VSDVPRDLEVVAATPTSLLISWHPPHPNIVSYHIYYGETGGNSPVfull-length with C-terminalQEFTVEGSKSTAKISGLKPGVDYTITVYAVAPEIEKYYQIWINYR P_(m)CX_(n1)CX_(n2)TP_(m)CX_(n1)CX_(n2) 16 ADX_4578_F03VSDVPRDLEVVAATPTSLLISWHPPHPNIVSYHIYYGETGGNSPVfull-length with C-terminalQEFTVEGSKSTAKISGLKPGVDYTITVYAVAPEIEKYYQIWINYRPmCX_(n1)CX_(n2) (m = 1, n1 = 5, n2 = 0) TPCPPPPPC 17 ADX_4578_F03VSDVPRDLEVVAATPTSLLISWHPPHPNIVSYHIYYGETGGNSPVfull-length with C-terminalQEFTVEGSKSTAKISGLKPGVDYTITVYAVAPEIEKYYQIWINYRPmCX_(n1)CX_(n2) (m = 1, n1 = 5, n2 = 0) TPCPPPPPCHHHHHH and His₆ tag 18ADX_4578_H08 EVVAATPTSLLISWSGYDYGDSYYRITYGETGGNSPVQEFTVPDG coreSNTATISGLKPGVDYTITVYAVEAYGKGYTRYTPISINYRT 19 ADX_4578_H08 SGYDYGDSYBC loop 20 ADX_4578_H08 PDGNST DE loop 21 ADX_4578_H08 VEAYGKGYTRYTPFG loop 22 ADX_4578_H08 VSDVPRDLEVVAATPTSLLISWSGYDYGDSYYRITYGETGGNSPVfull-length QEFTVPDGSNTATISGLKPGVDYTITVYAVEAYGKGYTRYTPISI NYRT 23ADX_4578_H08 MGVSDVPRDLEVVAATPTSLLISWSGYDYGDSYYRITYGETGGNSfull-length w/N-terminal leader andPVQEFTVPDGSNTATISGLKPGVDYTITVYAVEAYGKGYTRYTPI C-terminal tailSINYRTEIDKPSQ 24 ADX_4578_H08VSDVPRDLEVVAATPTSLLISWSGYDYGDSYYRITYGETGGNSPVfull-length with C-terminal PmXnQEFTVPDGSNTATISGLKPGVDYTITVYAVEAYGKGYTRYTPISI NYRTP_(m)X_(n) 25ADX_4578_H08 VSDVPRDLEVVAATPTSLLISWSGYDYGDSYYRITYGETGGNSPVfull-length with C-terminal PmCXnQEFTVPDGSNTATISGLKPGVDYTITVYAVEAYGKGYTRYTPISI NYRTP_(m)CX_(n) 26ADX_4578_H08 VSDVPRDLEVVAATPTSLLISWSGYDYGDSYYRITYGETGGNSPVfull-length with C-terminalQEFTVPDGSNTATISGLKPGVDYTITVYAVEAYGKGYTRYTPISI PmCXn (m = 1; n = 0)NYRTPC 27 ADX_4578_H08 VSDVPRDLEVVAATPTSLLISWSGYDYGDSYYRITYGETGGNSPVfull-length with C-terminalQEFTVPDGSNTATISGLKPGVDYTITVYAVEAYGKGYTRYTPISIPmCXn (m = 1; n = 0) and His₆ tag NYRTPCHHHHHH 28 ADX_4578_H08VSDVPRDLEVVAATPTSLLISWSGYDYGDSYYRITYGETGGNSPVfull-length with C-terminalQEFTVPDGSNTATISGLKPGVDYTITVYAVEAYGKGYTRYTPISI PmCX_(n1)CX_(n2)NYRTP_(m)CX_(n1)CX_(n2) 29 ADX_4578_H08VSDVPRDLEVVAATPTSLLISWSGYDYGDSYYRITYGETGGNSPVfull-length with C-terminalQEFTVPDGSNTATISGLKPGVDYTITVYAVEAYGKGYTRYTPISIPmCX_(n1)CX_(n2) (m = 1, n1 = 5, n2 = 0) NYRTPCPPPPPC 30 ADX_4578_H08VSDVPRDLEVVAATPTSLLISWSGYDYGDSYYRITYGETGGNSPVfull-length with C-terminalQEFTVPDGSNTATISGLKPGVDYTITVYAVEAYGKGYTRYTPISIPmCX_(n1)CX_(n2) (m = 1, n1 = 5, n2 = 0) NYRTPCPPPPPCHHHHHH and His₆ tag31 ADX_4578_B06 EVVAATPTSLLISWFPDRYVYYITYGETGGNSPVQEFTVEGHKQT coreAYISGLKPGVDYTITVYAAYISGLISINYRT 32 ADX_4578_B06 FPDRYV BC loop 33ADX_4578_B06 EGHKQT DE loop 34 ADX_4578_B06 AYISGL FG loop 35ADX_4578_B06 VSDVPRDLEVVAATPTSLLISWFPDRYVYYITYGETGGNSPVQEF full-lengthTVEGHKQTAYISGLKPGVDYTITVYAIYYYPDDFQGYPQPISINY RT 36 ADX_4578_B06MGVSDVPRDLEVVAATPTSLLISWFPDRYVYYITYGETGGNSPVQfull-length w/N-terminal leader andEFTVEGHKQTAYISGLKPGVDYTITVYAIYYYPDDFQGYPQPISI C-terminal tail NYRTEGSGS37 ADX_4578_B06 VSDVPRDLEVVAATPTSLLISWFPDRYVYYITYGETGGNSPVQEFfull-length with C-terminal PmXnTVEGHKQTAYISGLKPGVDYTITVYAIYYYPDDFQGYPQPISINY RTP_(m)X_(n) 38ADX_4578_B06 VSDVPRDLEVVAATPTSLLISWFPDRYVYYITYGETGGNSPVQEFfull-length with C-terminal PmCXnTVEGHKQTAYISGLKPGVDYTITVYAIYYYPDDFQGYPQPISINY RTP_(mC)X_(n) 39ADX_4578_B06 VSDVPRDLEVVAATPTSLLISWFPDRYVYYITYGETGGNSPVQEFfull-length with C-terminalTVEGHKQTAYISGLKPGVDYTITVYAIYYYPDDFQGYPQPISINY PmCXn (m = 1; n = 0) RTPC40 ADX_4578_B06 VSDVPRDLEVVAATPTSLLISWFPDRYVYYITYGETGGNSPVQEFfull-length with C-terminalTVEGHKQTAYISGLKPGVDYTITVYAIYYYPDDFQGYPQPISINYPmCXn (m = 1; n = 0) and His₆ tag RTPCHHHHHH 41 ADX_4578_B06VSDVPRDLEVVAATPTSLLISWFPDRYVYYITYGETGGNSPVQEFfull-length with C-terminalTVEGHKQTAYISGLKPGVDYTITVYAIYYYPDDFQGYPQPISINY PmCX_(n1)CX_(n2)RTP_(m)CX_(n1)CX_(n2) 42 ADX_4578_B06VSDVPRDLEVVAATPTSLLISWFPDRYVYYITYGETGGNSPVQEFfull-length with C-terminalTVEGHKQTAYISGLKPGVDYTITVYAIYYYPDDFQGYPQPISINYPmCX_(n1)CX_(n2) (m = 1, n1 = 5, n2 = 0) RTPCPPPPPC 43 ADX_4578_B06VSDVPRDLEVVAATPTSLLISWFPDRYVYYITYGETGGNSPVQEFfull-length with C-terminalTVEGHKQTAYISGLKPGVDYTITVYAIYYYPDDFQGYPQPISINYPmCX_(n1)CX_(n2) (m = 1, n1 = 5, n2 = 0) RTPCPPPPPCHHHHHH and His₆ tag44 ADX_4606_F06 EVVAATPTSLLISWNSGHSGQYYRITYGETGGNSPVQEFTVPRYG coreYTATISGLKPGVDYTITVYAVAHSEASAPISINYRT 45 ADX_4606_F06 NSGHSGQY BC loop 46ADX_4606_F06 PRYGYT DE loop 47 ADX_4606_F06 VAHSEASAP FG loop 48ADX_4606_F06 VSDVPRDLEVVAATPTSLLISWNSGHSGQYYRITYGETGGNSPVQ full-lengthEFTVPRYGYTATISGLKPGVDYTITVYAVAHSEASAPISINYRT 49 ADX_4606_F06MGVSDVPRDLEVVAATPTSLLISWNSGHSGQYYRITYGETGGNSPfull-length w/N-terminal leader andVQEFTVPRYGYTATISGLKPGVDYTITVYAVAHSEASAPISINYR C-terminal tail TEIDKPSQ50 ADX_4606_F06 VSDVPRDLEVVAATPTSLLISWNSGHSGQYYRITYGETGGNSPVQfull-length with C-terminal PmXnEFTVPRYGYTATISGLKPGVDYTITVYAVAHSEASAPISINYRTP _(m)X_(n) 51 ADX_4606_F06VSDVPRDLEVVAATPTSLLISWNSGHSGQYYRITYGETGGNSPVQfull-length with C-terminal PmCXnEFTVPRYGYTATISGLKPGVDYTITVYAVAHSEASAPISINYRTP _(m)CX_(n) 52 ADX_4606_F06VSDVPRDLEVVAATPTSLLISWNSGHSGQYYRITYGETGGNSPVQfull-length with C-terminalEFTVPRYGYTATISGLKPGVDYTITVYAVAHSEASAPISINYRTP PmCXn (m = 1; n = 0) C 53ADX_4606_F06 VSDVPRDLEVVAATPTSLLISWNSGHSGQYYRITYGETGGNSPVQfull-length with C-terminalEFTVPRYGYTATISGLKPGVDYTITVYAVAHSEASAPISINYRTPPmCXn (m = 1; n = 0)and His₆ tag CHHHHHH 54 ADX_4606_F06VSDVPRDLEVVAATPTSLLISWNSGHSGQYYRITYGETGGNSPVQfull-length with C-terminalEFTVPRYGYTATISGLKPGVDYTITVYAVAHSEASAPISINYRTP PmCX_(n1)CX_(n2)_(m)CX_(n1)CX_(n2) 55 ADX_4606_F06VSDVPRDLEVVAATPTSLLISWNSGHSGQYYRITYGETGGNSPVQfull-length with C-terminalEFTVPRYGYTATISGLKPGVDYTITVYAVAHSEASAPISINYRTPPmCX_(n1)CX_(n2) (m = 1, n1 = 5, n2 = 0) CPPPPPC 56 ADX_4606_F06VSDVPRDLEVVAATPTSLLISWNSGHSGQYYRITYGETGGNSPVQfull-length with C-terminalEFTVPRYGYTATISGLKPGVDYTITVYAVAHSEASAPISINYRTPPmCX_(n1)CX_(n2) (m = 1, n1 = 5, n2 = 0) CPPPPPCHHHHHH and His₆ tag 57ADX_5273_C01 EVVAATPTSLLISWSDPYEEERYYRITYGETGGNSPVQEFTVPAF coreHTTATISGLKPGVDYTITVYAVTYKHKYAYYYPPISINYRT 58 ADX_5273_C01 SDPYEEERYBC loop 59 ADX_5273_C01 PAFHTT DE loop 60 ADX_5273_C01 VTYKHKYAYYYPPFG loop 61 ADX_5273_C01 VSDVPRDLEVVAATPTSLLISWSDPYEEERYYRITYGETGGNSPVfull-length QEFTVPAFHTTATISGLKPGVDYTITVYAVTYKHKYAYYYPPISI NYRT 62ADX_5273_C01 MGVSDVPRDLEVVAATPTSLLISWSDPYEEERYYRITYGETGGNSfull-length w/N-terminal leader andPVQEFTVPAFHTTATISGLKPGVDYTITVYAVTYKHKYAYYYPPI C-terminal tailSINYRTEIDKPSQ 63 ADX_5273_C01VSDVPRDLEVVAATPTSLLISWSDPYEEERYYRITYGETGGNSPVfull-length with C-terminal PmXnQEFTVPAFHTTATISGLKPGVDYTITVYAVTYKHKYAYYYPPISI NYRTP_(m)X_(n) 64ADX_5273_C01 VSDVPRDLEVVAATPTSLLISWSDPYEEERYYRITYGETGGNSPVfull-length with C-terminal PmCXnQEFTVPAFHTTATISGLKPGVDYTITVYAVTYKHKYAYYYPPISI NYRTP_(m)CX_(n) 65ADX_5273_C01 VSDVPRDLEVVAATPTSLLISWSDPYEEERYYRITYGETGGNSPVfull-length with C-terminalQEFTVPAFHTTATISGLKPGVDYTITVYAVTYKHKYAYYYPPISI PmCXn (m = 1; n = 0)NYRTPC 66 ADX_5273_C01 VSDVPRDLEVVAATPTSLLISWSDPYEEERYYRITYGETGGNSPVfull-length with C-terminalQEFTVPAFHTTATISGLKPGVDYTITVYAVTYKHKYAYYYPPISIPmCXn (m = 1; n = 0) and His₆ tag NYRTPCHHHHHH 67 ADX_5273_C01VSDVPRDLEVVAATPTSLLISWSDPYEEERYYRITYGETGGNSPVfull-length with C-terminalQEFTVPAFHTTATISGLKPGVDYTITVYAVTYKHKYAYYYPPISI PmCX_(n1)CX_(n2)NYRTP_(m)CX_(n1)CX_(n2) 68 ADX_5273_C01VSDVPRDLEVVAATPTSLLISWSDPYEEERYYRITYGETGGNSPVfull-length with C-terminalQEFTVPAFHTTATISGLKPGVDYTITVYAVTYKHKYAYYYPPISIPmCX_(n1)CX_(n2) (m = 1, n1 = 5, n2 = 0) NYRTPCPPPPPC 69 ADX_5273_C01VSDVPRDLEVVAATPTSLLISWSDPYEEERYYRITYGETGGNSPVfull-length with C-terminalQEFTVPAFHTTATISGLKPGVDYTITVYAVTYKHKYAYYYPPISIPmCX_(n1)CX_(n2) (m = 1, n1 = 5, n2 = 0) NYRTPCPPPPPCHHHHHH and His₆ tag70 ADX_5273_D01 EVVAATPTSLLISWEPSYKDDRYYRITYGETGGNSPVQEFTVPSF coreHQTATISGLKPGVDYTITVYAVTYEPDEYYFYYPISINYRT 71 ADX_5273_D01 EPSYKDDRYBC loop 72 ADX_5273_D01 PSFHQT DE loop 73 ADX_5273_D01 VTYEPDEYYFYYPFG loop 74 ADX_5273_D01 VSDVPRDLEVVAATPTSLLISWEPSYKDDRYYRITYGETGGNSPVfull-length QEFTVPSFHQTATISGLKPGVDYTITVYAVTYEPDEYYFYYPISI NYRT 75ADX_5273_D01 MGVSDVPRDLEVVAATPTSLLISWEPSYKDDRYYRITYGETGGNSfull-length w/N-terminal leader andPVQEFTVPSFHQTATISGLKPGVDYTITVYAVTYEPDEYYFYYPI C-terminal tailSINYRTEIDKPSQ 76 ADX_5273_D01VSDVPRDLEVVAATPTSLLISWEPSYKDDRYYRITYGETGGNSPVfull-length with C-terminal PmXnQEFTVPSFHQTATISGLKPGVDYTITVYAVTYEPDEYYFYYPISI NYRTP_(m)X_(n) 77ADX_5273_D01 VSDVPRDLEVVAATPTSLLISWEPSYKDDRYYRITYGETGGNSPVfull-length with C-terminal PmCXnQEFTVPSFHQTATISGLKPGVDYTITVYAVTYEPDEYYFYYPISI NYRTP_(m)CX_(n) 78ADX_5273_D01 VSDVPRDLEVVAATPTSLLISWEPSYKDDRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPSFHQTATISGLKPGVDYTITVYAVTYEPDEYYFYYPISI PmCXn (m = 1; n = 0)NYRTPC 79 ADX_5273_D01 VSDVPRDLEVVAATPTSLLISWEPSYKDDRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPSFHQTATISGLKPGVDYTITVYAVTYEPDEYYFYYPISIPmCXn (m = 1; n = 0) and His₆ tag NYRTPCHHHHHH 80 ADX_5273_D01VSDVPRDLEVVAATPTSLLISWEPSYKDDRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPSFHQTATISGLKPGVDYTITVYAVTYEPDEYYFYYPISI PmCX_(n1)CX_(n2)NYRTP_(m)CX_(n1)CX_(n2) 81 ADX_5273_D01VSDVPRDLEVVAATPTSLLISWEPSYKDDRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPSFHQTATISGLKPGVDYTITVYAVTYEPDEYYFYYPISIPmCX_(n1)CX_(n2) (m = 1, n1 = 5, n2 = 0) NYRTPCPPPPPC 82 ADX_5273_D01VSDVPRDLEVVAATPTSLLISWEPSYKDDRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPSFHQTATISGLKPGVDYTITVYAVTYEPDEYYFYYPISIPmCX_(n1)CX_(n2) (m = 1, n1 = 5, n2 = 0) NYRTPCPPPPPCHHHHHH and His₆ tag83 ADX_5274_E01 EVVAATPTSLLISWSGDYHPHRYYRITYGETGGNSPVQEFTVPGE coreHETAATISGLKPGVDYTITVYAVTYDGEKADKYPPISINYRT 84 ADX_5274_E01 SGDYHPHRYBC loop 85 ADX_5274_E01 PGEHET DE loop 86 ADX_5274_E01 VTYDGEKADKYPPFG loop 87 ADX_5274_E01 VSDVPRDLEVVAATPTSLLISWSGDYHPHRYYRITYGETGGNSPVfull-length QEFTVPGEHETATISGLKPGVDYTITVYAVTYDGEKADKYPPISI NYRT 88ADX_5274 E01 MGVSDVPRDLEVVAATPTSLLISWSGDYHPHRYYRITYGETGGNSfull-length w/N-terminal leader andPVQEFTVPGEHETATISGLKPGVDYTITVYAVTYDGEKADKYPPI C-terminal tailSINYRTEIDKPSQ 89 ADX_5274_E01VSDVPRDLEVVAATPTSLLISWSGDYHPHRYYRITYGETGGNSPVfull-length w/C-terminal PmXnQEFTVPGEHETATISGLKPGVDYTITVYAVTYDGEKADKYPPISI NYRTP_(m)X_(n) 90ADX_5274_E01 VSDVPRDLEVVAATPTSLLISWSGDYHPHRYYRITYGETGGNSPVfull-length with C-terminal P_(m)CX_(n)QEFTVPGEHETATISGLKPGVDYTITVYAVTYDGEKADKYPPISI NYRTP_(m)CX_(n) 91ADC_5274_E01 EVVAATPTSLLISWSGDYHPHRYYRITYGETGGNSPVQEFTVPGEcore with PmCXn C-terminal HETATISGLKPGVDYTITVYAVTYDGEKADKYPPISINYRTPCmodification (m = 1; n = 0); aka ADX_6561_A01 core 92 ADX_5274_E01VSDVPRDLEVVAATPTSLLISWSGDYHPHRYYRITYGETGGNSPVfull-length with C-terminal P_(m)CX_(n)QEFTVPGEHETATISGLKPGVDYTITVYAVTYDGEKADKYPPISI (m = 1; n = 0) NYRTPC 93ADX_5274_E01 VSDVPRDLEVVAATPTSLLISWSGDYHPHRYYRITYGETGGNSPVfull-length with C-terminal P_(m)CX_(n)QEFTVPGEHETATISGLKPGVDYTITVYAVTYDGEKADKYPPISI(m = 1; n = 0) and His₆ tag NYRTPCHHHHHH 94 ADX_5274_A01MGVSDVPRDLEVVAATPTSLLISWSGDYHPHRYYRITYGETGGNSfull-length w/N-terminal leader, C-PVQEFTVPGEHETATISGLKPGVDYTITVYAVTYDGEKADKYPPIterminal P_(m)CX_(n) (m = 1; n = 0) and SINYRTPCHHHHHH His₆ tag 95ADX_5274_E01 VSDVPRDLEVVAATPTSLLISWSGDYHPHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHETATISGLKPGVDYTITVYAVTYDGEKADKYPPISI PmCXn₁CXn₂NYRTP_(m)CX_(n1)CX_(n2) 96 ADX_5274_E01VSDVPRDLEVVAATPTSLLISWSGDYHPHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHETATISGLKPGVDYTITVYAVTYDGEKADKYPPISIPmCXn₁CXn₂ (m = 1; n1 = 5; n2 = 0) NYRTPCPPPPPC 97 ADX_5274_E01VSDVPRDLEVVAATPTSLLISWSGDYHPHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHETATISGLKPGVDYTITVYAVTYDGEKADKYPPISIPmCXn₁CXn₂ (m = 1; n1 = 5; n2 = 0) NYRTPCPPPPPCHHHHHH and His₆ tag 98ADX_6077_A01 EVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGE coreHVTATISGLKPGVDYTITVYAVTYDGEKAATDWSISINYRT 99 ADX_6077_F02 SDDYHAHRYBC loop 100 ADX_6077_F02 PGEHVT DE loop 101 ADX_6077_F02 VTYDGEKAATDWSFG loop 102 ADX_6077_F02 VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length QEFTVPGEHVTATISGLKPGVDYTITVYAVTYDGEKAATDWSISI NYRT 103ADX_6077_F02 VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminal tailQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDGEKAATDWSISI NYRTEIEKPCQ 104ADX_6077_F02 GVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDGEKAATDWSIS (G) INYRT 105 ADX_6077_F02MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDGEKAATDWSI (MG) SINYRT 106ADX_6077_F02 EVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal PmXnHVTATISGLKPGVDYTITVYAVTYDGEKAATDWSISINYRTP_(m)X_(n) 107 ADX_6077_F02VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminal PmXnQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDGEKAATDWSISI NYRTP_(m)X_(n) 108ADX_6077_F02 GVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDGEKAATDWSIS (G) and C-terminal PmXnINYRTP_(m)X_(n) 109 ADX_6077_F02MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDGEKAATDWSI (MG) and C-terminal PmXnSINYRTP_(m)X_(n) 110 ADX_6077_F02EVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGE core with C-terminal PmCXnHVTATISGLKPGVDYTITVYAVTYDGEKAATDWSISINYRTP_(m)CX_(n) 111 ADX_6077_F02VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminal P_(m)CX_(n)QEFTVPGEHVTATISGLKPGVDYTITVYAVTYDGEKAATDWSISI NYRTP_(m)CX_(n) 112ADX_6077_F02 GVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDGEKAATDWSIS(G) and C-terminal P_(m)CX_(n) INYRTP_(m)CX_(n) 113 ADX_6077_F02MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDGEKAATDWSI(MG) and C-terminal P_(m)CX_(n) SINYRTP_(m)CX_(n) 114 ADX_6077_F02EVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal PmCXn (m = 1;HVTATISGLKPGVDYTITVYAVTYDGEKAATDWSISINYRTPC n = 0) 115 ADX_6077_F02VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminal PmCXnQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDGEKAATDWSISI (M = 1; n = 0) NYRTPC 116ADX_6007_F02 GVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length w/N-terminal leader (G)VQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDGEKAATDWSIS and PmCXn C-terminalINYRTPC modification (m = 1; n = 0) 117 ADX_6077_F02MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length w/N-terminal eaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDGEKAATDWSI (MG) and PmCXn C-terminalSINYRTPC modification (m = 1; n = 0) 118 ADX_6077_F02MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length w/PmCXn C-terminalPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDGEKAATDWSI modificationSINYRTPCHHHHHH (m = 1; n = 0) and His₆ tag 119 ADX_6077_F02EVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal PmCXn₁CXn₂HVTATISGLKPGVDYTITVYAVTYDGEKAATDWSISINYRTP_(m)CXn ₁CX_(n2) 120ADX_6077_F02 VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDGEKAATDWSISI PmCXn₁CXn₂NYRTP_(m)CX_(n1)CX_(n2) 121 ADX_6077_F02GVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDGEKAATDWSIS(G), and C-terminal PmCXn₁CXn₂ INYRTP_(m)CX_(n1)CX_(n2) 122 ADX_6077_F02MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDGEKAATDWSI (MG), and C-terminalSINYRTP_(m)CX_(n1)CX_(n2) PmCXn₁CXn₂ 123 ADX_6077_F02EVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal PmCXn₁CXn₂HVTATISGLKPGVDYTITVYAVTYDGEKAATDWSISINYRTPCPP (m = 1; n1 = 5; n2 = 0)PPPC 124 ADX_6077_F02 VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDGEKAATDWSISIPmCXn₁Xn₂ (m = 1; n1 = 5, n2 = 0) NYRTPCPPPPPC 125 ADX_6077_F02GVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDGEKAATDWSIS(G), and C-terminal PmCXn₁CXn₂ INYRTPCPPPPPC (m = 1; n1 = 5, n2 = 0) 126ADX_6077_F02 MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length w/N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDGEKAATDWSI (MG), and C-terminalSINYRTPCPPPPPC PmCXn₁CXn₂ (m = 1; n1 = 5, n2 = 0) 127 ADX_6077_F02MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNS full-length w/C-terminalPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDGEKAATDWSI PmCXn₁CXn₂ (m = 1; n1 = 5,SINYRTPCPPPPPCHHHHHH n2 = 0) and His₆ tag 128 ADX_6077_F02 DG→EG mutantEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGE coreHVTATISGLKPGVDYTITVYAVTYEGEKAATDWSISINYRT 129 ADX_6077_F02 DG→EG mutantVTYEGEKAATDWS FG loop 130 ADX_6077_F02 DG→EG mutantVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPV full-lengthQEFTVPGEHVTATISGLKPGVDYTITVYAVTYEGEKAATDWSISI NYRT 131ADX_6077_F02 DG→EG mutant GVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYEGEKAATDWSIS (G) INYRT 132ADX_6077_F02 DG→EG mutant MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYEGEKAATDWSI (MG) SINYRT 133ADX_6077_F02 DG→EG mutant EVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal PmXnHVTATISGLKPGVDYTITVYAVTYEGEKAATDWSISINYRTP_(m)X_(n) 134ADX_6077_F02 DG→EG mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminal PmXnQEFTVPGEHVTATISGLKPGVDYTITVYAVTYEGEKAATDWSISI NYRTP_(m)X_(n) 135ADX_6077_F02 DG→EG mutant GVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYEGEKAATDWSIS (G) and C-terminal PmXnINYRTP_(m)X_(n) 136 ADX_6077_F02 DG→EG mutantMGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYEGEKAATDWSI (MG) and C-terminal PmXnSINYRTP_(m)X_(n) 137 ADX_6077_F02 DG→EG mutantEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGE core with C-terminal PmCXnHVTATISGLKPGVDYTITVYAVTYEGEKAATDWSISINYRTP_(m)CX_(n) 138ADX_6077_F02 DG→EG mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminal PmCXnQEFTVPGEHVTATISGLKPGVDYTITVYAVTYEGEKAATDWSISI NYRTP_(m)CX_(n) 139ADX_6077_F02 DG→EG mutant GVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYEGEKAATDWSIS(G) and C-terminal P_(m)CX_(n) INYRTP_(m)CX_(n) 140ADX_6077_F02 DG→EG mutant MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYEGEKAATDWSI(MG) and C-terminal P_(m)CX_(n) SINYRTP_(m)CX_(n) 141ADX_6077_F02 DG→EG mutant EVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal PmCXn (m = 1;HVTATISGLKPGVDYTITVYAVTYEGEKAATDWSISINYRTPC n = 0) 142ADX_6077_F02 DG→EG mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYEGEKAATDWSISI PmCXn (m = 1; n = 0)NYRTPC 143 ADX_6077_F02 DG→EG mutantGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length w/N-terminal leader (G)VQEFTVPGEHVTATISGLKPGVDYTITVYAVTYEGEKAATDWSIS and PmCXn C-terminalINYRTPC modification (m = 1; n = 0) 144 ADX_6077_F02 DG→EG mutantMGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length w/N-terminal eaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYEGEKAATDWSI (MG) and PmCXn C-terminalSINYRTPC modification (m = 1; n = 0) 145 ADX_6077_F02 DG→EG mutantVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYEGEKAATDWSISIPmCXn (m = 1; n = 0) and His₆ tag NYRTPCHHHHHH 146ADX_6077_F02 DG→EG mutant EVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal PmCXn₁CXn₂HVTATISGLKPGVDYTITVYAVTYEGEKAATDWSISINYRTP_(m)CX_(n) ₁CX₂ 147ADX_6077_F02 DG→EG mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminal QEFTVPGEHVTATISGLKPGVDYTITVYAVTYEGEKAATDWSISI P^(m)CXn₁CX₂NYRTP_(m)CX_(n1)CX₂ 148 ADX_6077_F02 DG→EG mutantGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYEGEKAATDWSIS(G), and C-terminal PmCXn₁CXn₂ INYRTP_(m)CX_(n1)CX₂ 149ADX_6077_F02 DG→EG mutant MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYEGEKAATDWSI (MG), and C-terminalSINYRTP_(m)CX_(n1)CX₂ PmCXn₁CXn₂ 150 ADX_6077_F02 DG→EG mutantEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal PmCXn₁CXn₂HVTATISGLKPGVDYTITVYAVTYEGEKAATDWSISINYRTPCPP (m = 1; n1 = 5; n2 = 0)PPPC 151 ADX_6077_F02 DG→EG mutantVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYEGEKAATDWSISIPmCXn1CXn2 (m = 1; n1 = 5; n2 = 0) NYRTPCPPPPPC 152ADX_6077_F02 DG→EG mutant GVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPMGGEHVTATISGLKPGVDYTITVYAVTYEGEKAATDWS(G), and C-terminal PmCXn₁CXn₂ ISINYRTPCPPPPPC (m = 1; n1 = 5, n2 = 0)153 ADX_6077_F02 DG→EG mutantMGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYEGEKAATDWSI (MG), and C-terminalSINYRTPCPPPPPC PmCXn₁CXn₂ (m = 1; n1 = 5, n2 = 0) 154ADX_6077_F02 DG→EG mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYEGEKAATDWSISIP_(m)CX_(n1)CX_(n2) (m = 1; n1 = 5; n2 = 0), NYRTPCPPPPPCHHHHHHand His₆ tag 155 ADX_6077_F02 DG→SG mutantEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGE coreHVTATISGLKPGVDYTITVYAVTYSGEKAATDWSISINYRT 156 ADX_6077_F02 DG→SG mutantVTYSGEKAATDWS FG loop 157 ADX_6077_F02 DG→SG mutantVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPV full-lengthQEFTVPGEHVTATISGLKPGVDYTITVYAVTYSGEKAATDWSISI NYRT 158ADX_6077_F02 DG→SG mutant GVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYSGEKAATDWSIS (G) INYRT 159ADX_6077_F02 DG→SG mutant MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYSGEKAATDWSI (MG) SINYRT 160ADX_6077_F02 DG→SG mutant EVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal PmXnHVTATISGLKPGVDYTITVYAVTYSGEKAATDWSISINYRTP_(m)X_(n) 161ADX_6077_F02 DG→SG mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminal PmXnQEFTVPGEHVTATISGLKPGVDYTITVYAVTYSGEKAATDWSISI NYRTP_(m)X_(n) 162ADX_6077_F02 DG→SG mutant GVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYSGEKAATDWSIS (G) and C-terminal PmXnINYRTP_(m)X_(n) 163 ADX_6077_F02 DG→SG mutantMGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYSGEKAATDWSI (MG) and C-terminal PmXnSINYRTP_(m)X_(n) 164 ADX_6077_F02 DG→SG mutantEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal P_(m)CX_(n)HVTATISGLKPGVDYTITVYAVTYSGEKAATDWSISINYRTPmCXn 165ADX_6077_F02 DG→SG mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminal PmCXnQEFTVPGEHVTATISGLKPGVDYTITVYAVTYSGEKAATDWSISI NYRTP_(m)CX_(n) 166ADX_6077_F02 DG→SG mutant GVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYSGEKAATDWSIS(G) and C-terminal P_(m)CX_(n) INYRTP_(m)CX_(n) 167 ADX_6077_F02 DG→SGMGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYSGEKAATDWSI(MG) and C-terminal P_(m)CX_(n) SINYRTP_(m)CX_(n) 168ADX_6077_F02 DG→SG mutant EVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal PmCXn (m = 1;HVTATISGLKPGVDYTITVYAVTYSGEKAATDWSISINYRTPC n = 0) 169ADX_6077_F02 DG→SG mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYSGEKAATDWSISI PmCXn (m = 1; n = 0)NYRTPC 170 ADX_6077_F02 DG→SG mutantGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length w/N-terminal leader andVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYSGEKAATDWSIS PmCXn (m = 1; n = 0)INYRTPC 171 ADX_6077_F02 DG→SG mutantMGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length w/N-terminal eaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYSGEKAATDWSI (MG) and PmCXn C-terminalSINYRTPC modification (m = 1; n = 0) 172 ADX_6077_F02 DG→SG mutantVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYSGEKAATDWSISIPmCXn (m = 1; n = 0) and His₆ tag NYRTPCHHHHHH 173ADX_6077_F02 DG→SG mutant EVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal PmCXn₁CXn₂HVTATISGLKPGVDYTITVYAVTYSGEKAATDWSISINYRTP_(m)CX_(n) ₁CX₂ 174ADX_6077_F02 DG→SG mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYSGEKAATDWSISI P_(m)CX_(n1)CX_(n2)NYRTP_(m)CX_(n1)CX₂ 175 ADX_6077_F02 DG→SG mutantGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYSGEKAATDWSIS(G), and C-terminal PmCXn₁CXn₂ INYRTP_(m)CX_(n1)CX₂ 176ADX_6077_F02 DG→SG mutant MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYSGEKAATDWSI (MG), and C-terminalSINYRTP_(m)CX_(n1)CX₂ PmCXn₁CXn₂ 177 ADX_6077_F02 DG→SG mutantEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal PmCXn₁CXn₂HVTATISGLKPGVDYTITVYAVTYSGEKAATDWSISINYRTPCPP (m = 1; n1 = 5; n2 = 0)PPPC 178 ADX_6077_F02 DG→SG mutantVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYSGEKAATDWSISIP_(m)CX_(n1)CX_(n2) (m = 1; n1 = 5; n2 = 0) NYRTPCPPPPPC 179ADX_6077_F02 DG→SG mutant GVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYSGEKAATDWSIS(G), and C-terminal PmCXn₁CXn₂ INYRTPCPPPPPC (m = 1; n1 = 5, n2 = 0) 180ADX_6077_F02 DG→SG mutant MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYSGEKAATDWSI (MG), and C-terminalSINYRTPCPPPPPC PmCXn₁CXn₂ (m = 1; n1 = 5, n2 = 0) 181ADX_6077_F02 DG→SG mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYSGEKAATDWSISIP_(m)CX_(n1)CX_(n2) (m = 1 ; n1 = 5; n2 = 0), NYRTPCPPPPPCHHHHHHand His₆ tag 182 ADX_6077_F02 DG→AG mutantEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGE coreHVTATISGLKPGVDYTITVYAVTYAGEKAATDWSISINYRT 183 ADX_6077_F02 DG→AG mutantVTYAGEKAATDWS FG loop 184 ADX_6077_F02 DG→AG mutantVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPV full-lengthQEFTVPGEHVTATISGLKPGVDYTITVYAVTYAGEKAATDWSISI NYRT 185ADX_6077_F02 DG→AG mutant GVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYAGEKAATDWSIS (G) INYRT 186ADX_6077_F02 DG→AG mutant MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYAGEKAATDWSI (MG) SINYRT 187ADX_6077_F02 DG→AG mutant LEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGcore with C-terminal PmXnEHVTATISGLKPGVDYTITVYAVTYAGEKAATDWSISINYRTP_(m)X_(n) 188ADX_6077_F02 DG→AG mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminal PmXnQEFTVPGEHVTATISGLKPGVDYTITVYAVTYAGEKAATDWSISI NYRTP_(m)X_(n) 189ADX_6077_F02 DG→AG mutant GVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYAGEKAATDWSIS (G) and C-terminal PmXnINYRTP_(m)X_(n) 190 ADX_6077_F02 DG→AG mutantMGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYAGEKAATDWSI (MG) and C-terminal PmXnSINYRTP_(m)X_(n) 191 ADX_6077_F02 DG→AG mutantEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal P_(m)CX_(n)HVTATISGLKPGVDYTITVYAVTYAGEKAATDWSISINYRTP_(m)CX_(n) 192ADX_6077_F02 DG→AG mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminal PmCXnQEFTVPGEHVTATISGLKPGVDYTITVYAVTYAGEKAATDWSISI NYRTP_(m)CX_(n) 193ADX_6077_F02 DG→AG mutant GVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYAGEKAATDWSIS(G) and C-terminal P_(m)CX_(n) INYRTP_(m)CX_(n) 194ADX_6077_F02 DG→AG mutant MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYAGEKAATDWSI(MG) and C-terminal P_(m)CX_(n) SINYRTP_(m)CX_(n) 195ADX_6077_F02 DG→AG mutant EVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal PmCXn (m = 1;HVTATISGLKPGVDYTITVYAVTYAGEKAATDWSISINYRTPC n = 0) 196ADX_6077_F02 DG→AG mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYAGEKAATDWSISI PmCXn (m = 1; n = 0)NYRTPC 197 ADX_6077_F02 DG→AG mutantGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length w/N-terminal leader (G)VQEFTVPGEHVTATISGLKPGVDYTITVYAVTYAGEKAATDWSIS and PmCXn C-terminalINYRTPC modification (m = 1; n = 0) 198 ADX_6077_F02 DG→AG mutantMGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length w/N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYAGEKAATDWSI (MG) and PmCXn C-terminalSINYRTPC modification (m = 1; n = 0) 199 ADX_6077_F02 DG→AG mutantVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYAGEKAATDWSISIPmCXn (m = 1; n = 0) and His₆ tag NYRTPCHHHHHH 200ADX_6077_F02 DG→AG mutant EVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal PmCXn₁CXn₂HVTATISGLKPGVDYTITVYAVTYAGEKAATDWSISINYRTP_(m)CX_(n) ₁CX₂ 201ADX_6077_F02 DG→AG mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYAGEKAATDWSISI P_(m)CXn₁CX₂NYRTP_(m)CX_(n1)CX₂ 202 ADX_6077_F02 DG→AG mutantGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYAGEKAATDWSIS(G), and C-terminal PmCXn₁CXn₂ INYRTP_(m)CX_(n1)CX₂ 203ADX_6077_F02 DG→AG mutant MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYAGEKAATDWSI (MG), and C-terminalSINYRTP_(m)CX_(n1)CX₂ PmCXn₁CXn₂ 204 ADX_6077_F02 DG→AG mutantEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal PmCXn₁CXn₂HVTATISGLKPGVDYTITVYAVTYAGEKAATDWSISINYRTPCPP (m = 1; n1 = 5; n2 = 0)PPPC 205 ADX_6077_F02 DG→AG mutantVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYAGEKAATDWSISIP_(m)CX_(n1)CX_(n2) (m = 1; n1 = 5; n2 = 0) NYRTPCPPPPPC 206ADX_6077_F02 DG→AG mutant GVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYAGEKAATDWSIS(G), and C-terminal PmCXn₁CXn₂ INYRTPCPPPPPC (m = 1; n1 = 5, n2 = 0) 207ADX_6077_F02 DG→AG mutant MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYAGEKAATDWSI (MG), and C-terminalSINYRTPCPPPPPC PmCXn₁CXn₂ (m = 1; n1 = 5, n2 = 0) 208ADX_6077_F02 AG→AG mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYAGEKAATDWSISIP_(m)CX_(n1)CX_(n2) (m = 1 ; n1 = 5; n2 = 0), NYRTPCPPPPPCHHHHHHand His₆ tag 209 ADX_6077 F02 DG→GG mutantEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGE coreHVTATISGLKPGVDYTITVYAVTYGGEKAATDWSISINYRT 210 ADX_6077_F02 DG→GG mutantTYGGEKAATDWS FG loop 211 ADX_6077_F02 DG→GG mutantVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPV full-lengthQEFTVPGEHVTATISGLKPGVDYTITVYAVTYGGEKAATDWSISI NYRT 212ADX_6077_F02 DG→GG mutant GVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYGGEKAATDWSIS (G) INYRT 213ADX_6077_F02 DG→GG mutant MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYGGEKAATDWSI (MG) SINYRT 214ADX_6077_F02 DG→GG mutant EVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal PmXnHVTATISGLKPGVDYTITVYAVTYGGEKAATDWSISINYRTP_(m)X_(n) 215ADX_6077_F02 DG→GG mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminal PmXnQEFTVPGEHVTATISGLKPGVDYTITVYAVTYGGEKAATDWSISI NYRTP_(m)X_(n) 216ADX_6077_F02 DG→GG mutant GVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYGGEKAATDWSIS (G) and C-terminal PmXnINYRTP_(m)X_(n) 217 ADX_6077_F02 DG→GG mutantMGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYGGEKAATDWSI (MG) and C-terminal PmXnSINYRTP_(m)X_(n) 218 ADX_6077_F02 DG→GG mutantEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal P_(m)CX_(n)HVTATISGLKPGVDYTITVYAVTYGGEKAATDWSISINYRTP_(m)CX_(n) 219ADX_6077_F02 DG→GG mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminal PmCXnQEFTVPGEHVTATISGLKPGVDYTITVYAVTYGGEKAATDWSISI NYRTP_(m)CX_(n) 220ADX_6077_F02 DG→GG mutant MVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYGGEKAATDWSIS(G) and C-terminal P_(m)CX_(n) INYRTP_(m)CX_(n) 221ADX_6077_F02 DG→GG mutant MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYGGEKAATDWSI(MG) and C-terminal P_(m)CX_(n) SINYRTP_(m)CX_(n) 222ADX_6077_F02 DG→GG mutant EVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal PmCXn (m = 1;HVTATISGLKPGVDYTITVYAVTYGGEKAATDWSISINYRTPC n = 0) 223ADX_6077_F02 DG→GG mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYGGEKAATDWSISI PmCXn (m = 1; n = 0)NYRTPC 224 ADX_6077_F02 DG→GG mutantGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYGGEKAATDWSIS(G) and C-terminal P_(m)CX_(n) INYRTPC 225 ADX_6077_F02 DG→GG mutantMGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSFull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYGGEKAATDWSI(MG) and C-terminal P_(m)CX_(n) SINYRTPC 226 ADX_6077_F02 DG→GG mutantVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYGGEKAATDWSISIPmCXn (m = 1; n = 0) and His₆ tag NYRTPCHHHHHH 227ADX_6077_F02 DG→GG mutant EVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal PmCXn₁CXn₂HVTATISGLKPGVDYTITVYAVTYGGEKAATDWSISINYRTP_(m)CX_(n) ₁CX₂ 228ADX_6077_F02 DG→GG mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYGGEKAATDWSISI P_(m)CX_(n1)CX₂NYRTP_(m)CX_(n1)CX₂ 229 ADX_6077_F02 DG→GG mutantGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYGGEKAATDWSIS(G), and C-terminal PmCXn₁CXn₂ INYRTP_(m)CX_(n1)CX₂ 230ADX_6077_F02 DG→GG mutant MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYGGEKAATDWSI (MG), and C-terminalSINYRTP_(m)CX_(n1)CX₂ PmCXn₁CXn₂ 231 ADX_6077_F02 DG→GG mutantEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal PmCXn₁CXn₂HVTATISGLKPGVDYTITVYAVTYGGEKAATDWSISINYRTPCPP (m = 1; n1 = 5; n2 = 0)PPPC 232 ADX_6077_F02 DG→GG mutantVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYGGEKAATDWSISIP_(m)CX_(n1)CX_(n2) (m = 1; n1 = 5; n2 = 0) NYRTPCPPPPPC 233ADX_6077_F02 DG→GG mutant GVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYGGEKAATDWSIS(G), and C-terminal PmCXn₁CXn₂ INYRTPCPPPPPC (m = 1; n1 = 5, n2 = 0) 234ADX_6077_F02 DG→GG mutant MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYGGEKAATDWSI (MG), and C-terminalSINYRTPCPPPPPC PmCXn₁CXn₂ (m = 1; n1 = 5, n2 = 0) 235ADX_6077_F02 DG→GG mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYGGEKAATDWSISIP_(m)CX_(n1)CX_(n2) (m = 1 ; n1 = 5; n2 = 0), NYRTPCPPPPPCHHHHHHand His₆ tag 236 ADX_6077_F02 DG→DS mutantEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGE coreHVTATISGLKPGVDYTITVYAVTYDSEKAATDWSISINYRT 237 ADX_6077_F02 DG→DS mutantVTYDSEKAATDWS FG loop 238 ADX_6077_F02 DG→DS mutantVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPV full-lengthQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDSEKAATDWSISI NYRT 239ADX_6077_F02 DG→DS mutant GVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDSEKAATDWSIS (G) INYRT 240ADX_6077_F02 DG→DS mutant MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDSEKAATDWSI (MG) SINYRT 241ADX_6077_F02 DG→DS mutant EVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal PmXnHVTATISGLKPGVDYTITVYAVTYDSEKAATDWSISINYRTP_(m)X_(n) 242ADX_6077_F02 DG→DS mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminal PmXnQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDSEKAATDWSISI NYRTP_(m)X_(n) 243ADX_6077_F02 DG→DS mutant GVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDSEKAATDWSIS (G) and C-terminal PmXnINYRTP_(m)X_(n) 244 ADX_6077_F02 DG→DS mutantMGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDSEKAATDWSI (MG) and C-terminal PmXnSINYRTP_(m)X_(n) 245 ADX_6077_F02 DG→DS mutantEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal P_(m)CX_(n)HVTATISGLKPGVDYTITVYAVTYDSEKAATDWSISINYRTP_(m)CX_(n) 246ADX_6077_F02 DG→DS mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminal PmCXnQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDSEKAATDWSISI NYRTP_(m)CX_(n) 247ADX_6077_F02 DG→DS mutant GVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDSEKAATDWSIS(G) and C-terminal P_(m)CX_(n) INYRTP_(m)CX_(n) 248ADX_6077_F02 DG→DS mutant MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDSEKAATDWSI(MG) and C-terminal P_(m)CX_(n) SINYRTP_(m)CX_(n) 249ADX_6077_F02 DG→DS mutant EVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal PmCXn (m = 1;HVTATISGLKPGVDYTITVYAVTYDSEKAATDWSISINYRTPC n = 0) 250ADX_6077_F02 DG→DS mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDSEKAATDWSISI PmCXn (m = 1; n = 0)NYRTPC 251 ADX_6077_F02 DG→DS mutantGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length w/ N-terminal leader (G)VQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDSEKAATDWSIS and PmCXn C-terminalINYRTPC modification (m = 1; n = 0) 252 ADX_6077_F02 DG→DS mutantMGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length w/N-terminal eaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDSEKAATDWSI (MG) and PmCXn C-terminalSINYRTPC modification (m = 1; n = 0) 253 ADX_6077_F02 DG→DS mutantVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDSEKAATDWSISIPmCXn (m = 1; n = 0) and His₆ tag NYRTPCHHHHHH 254ADX_6077_F02 DG→DS mutant EVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal PmCXn₁CXn₂HVTATISGLKPGVDYTITVYAVTYDSEKAATDWSISINYRTP_(m)CX_(n) ₁CX₂ 255ADX_6077_F02 DG→DS mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDSEKAATDWSISI P_(m)CX_(n1)CX₂NYRTP_(m)CX_(n1)CX₂ 256 ADX_6077_F02 DG→DS mutantGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDSEKAATDWSIS(G), and C-terminal PmCXn₁CXn₂ INYRTP_(m)CX_(n1)CX₂ 257ADX_6077_F02 DG→DS mutant MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDSEKAATDWSI (MG), and C-terminalSINYRTP_(m)CX_(n1)CX₂ PmCXn₁CXn₂ 258 ADX_6077_F02 DG→DS mutantEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal PmCXn₁CXn₂HVTATISGLKPGVDYTITVYAVTYDSEKAATDWSISINYRTPCPP (m = 1; n1 = 5; n2 = 0)PPPC 259 ADX_6077_F02 DG→DS mutantVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDSEKAATDWSISIP_(m)CX_(n1)CX_(n2) (m = 1; n1 = 5; n2 = 0) NYRTPCPPPPPC 260ADX_6077_F02 DG→DS mutant GVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDSEKAATDWSIS(G), and C-terminal PmCXn₁CXn₂ INYRTPCPPPPPC (m = 1; n1 = 5, n2 = 0) 261ADX_6077_F02 DG→DS mutant MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDSEKAATDWSI (MG), and C-terminalSINYRTPCPPPPPC PmCXn₁CXn₂ (m = 1; n1 = 5, n2 = 0) 262ADX_6077_F02 DG→DS mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDSEKAATDWSISIP_(m)CX_(n1)CX_(n2) (m = 1 ; n1 = 5; n2 = 0), NYRTPCPPPPPCHHHHHHand His₆ tag 263 ADX_6077_F02 DG→DA mutantEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGE coreHVTATISGLKPGVDYTITVYAVTYDAEKAATDWSISINYRT 264 ADX_6077 F02 DG→DA mutantVTYDAEKAATDWS FG loop 265 ADX_6077_F02 DG→DA mutantVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPV full-lengthQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDAEKAATDWSISI NYRT 266ADX_6077_F02 DG→DA mutant GVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDAEKAATDWSIS (G) INYRT 267ADX_6077_F02 DG→DA mutant MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDAEKAATDWSI (MG) SINYRT 268ADX_6077_F02 DG→DA mutant EVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal PmXnHVTATISGLKPGVDYTITVYAVTYDAEKAATDWSISINYRTP_(m)X_(n) 269 ADX_6077_F02VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPV DG→DA mutantQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDAEKAATDWSISIfull-length with C-terminal PmXn NYRTP_(m)X_(n) 270ADX_6077_F02 DG→DA mutant GVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDAEKAATDWSIS (G) and C-terminal PmXnINYRTP_(m)X_(n) 271 ADX_6077_F02 DG→DA mutantMGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDAEKAATDWSI (MG) and C-terminal PmXnSINYRTP_(m)X_(n) 272 ADX_6077_F02 DG→DA mutantEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal P_(m)CX_(n)HVTATISGLKPGVDYTITVYAVTYDAEKAATDWSISINYRTP_(m)CX_(n) 273ADX_6077_F02 DG→DA mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminal PmCXnQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDAEKAATDWSISI NYRTP_(m)CX_(n) 274ADX_6077_F02 DG→DA mutant GVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDAEKAATDWSIS(G) and C-terminal P_(m)CX_(n) INYRTP_(m)CX_(n) 275ADX_6077_F02 DG→DA mutant MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDAEKAATDWSI(MG) and C-terminal P_(m)CX_(n) SINYRTP_(m)CX_(n) 276ADX_6077_F02 DG→DA mutant EVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal PmCXn (m = 1;HVTATISGLKPGVDYTITVYAVTYDAEKAATDWSISINYRTPC n = 0) 277ADX_6077_F02 DG→DA mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDAEKAATDWSISI PmCXn (m = 1; n = 0)NYRTPC 278 ADX_6077_F02 DG→DA mutantGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length w/N-terminal leader andVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDAEKAATDWSISPmCXn C-terminal modification INYRTPC (m = 1; n = 0) 279ADX_6077_F02 DG→DA mutant MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length w/N-terminal eaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDAEKAATDWSI (MG) and PmCXn C-terminalSINYRTPC modification (m = 1; n = 0) 280 ADX_6077_F02 DG→DA mutantVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDAEKAATDWSISIPmCXn (m = 1; n = 0) and His₆ tag NYRTPCHHHHHH 281ADX_6077_F02 DG→DA mutant EVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal PmCXn₁CXn₂HVTATISGLKPGVDYTITVYAVTYDAEKAATDWSISINYRTP_(m)CX_(n) ₁CX₂ 282ADX_6077_F02 DG→DA mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDAEKAATDWSISI P_(m)CX_(n1)CX₂NYRTP_(m)CX_(n1)CX₂ 283 ADX_6077_F02 DG→DA mutantGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDAEKAATDWSIS(G), and C-terminal PmCXn₁CXn₂ INYRTP_(m)CX_(n1)CX₂ 284ADX_6077_F02 DG→DA mutant MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDAEKAATDWSI (MG), and C-terminalSINYRTP_(m)CX_(n1)CX₂ PmCXn₁CXn₂ 285 ADX_6077_F02 DG→DA mutantEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal PmCXn₁CXn₂HVTATISGLKPGVDYTITVYAVTYDAEKAATDWSISINYRTPCPP (m = 1; n1 = 5; n2 = 0)PPPC 286 ADX_6077_F02 DG→DA mutantVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDAEKAATDWSISIP_(m)CX_(n1)CX_(n2) (m = 1; n1 = 5; n2 = 0) NYRTPCPPPPPC 287ADX_6077_F02 DG→DA mutant GVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDAEKAATDWSIS(G), and C-terminal PmCXn₁CXn₂ INYRTPCPPPPPC (m = 1; n1 = 5, n2 = 0) 288ADX_6077_F02 DG→DA mutant MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDAEKAATDWSI (MG), and C-terminalSINYRTPCPPPPPC PmCXn₁CXn₂ (m = 1; n1 = 5, n2 = 0) 289ADX_6077_F02 DG→DA mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDAEKAATDWSISIP_(m)CX_(n1)CX_(n2) (m = 1; n1 = 5; n2 = 0), NYRTPCPPPPPCHHHHHHand His₆ tag 290 ADX_6077_F02 DG→DL mutantEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGE coreHVTATISGLKPGVDYTITVYAVTYDLEKAATDWSISINYRT 291 ADX_6077_F02 DG→DL mutantVTYDLEKAATDWS FG loop 292 ADX_6077_F02 DG→DL mutantVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPV full-lengthQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDLEKAATDWSISI NYRT 293ADX_6077_F02 DG→DL mutant GVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDLEKAATDWSIS (G) INYRT 294ADX_6077_F02 DG→DL mutant MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDLEKAATDWSI (MG) SINYRT 295ADX_6077_F02 DG→DL mutant EVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal PmXnHVTATISGLKPGVDYTITVYAVTYDLEKAATDWSISINYRTP_(m)X_(n) 296ADX_6077_F02 DG→DL mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminal PmXnQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDLEKAATDWSISI NYRTP_(m)X_(n) 297ADX_6077_F02 DG→DL mutant MVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDLEKAATDWSIS (G) and C-terminal PmXnINYRTP_(m)X_(n) 298 ADX_6077_F02 DG→DL mutantMGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDLEKAATDWSI (MG) and C-terminal PmXnSINYRTP_(m)X_(n) 299 ADX_6077_F02 DG→DL mutantEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal P_(m)CX_(n)HVTATISGLKPGVDYTITVYAVTYDLEKAATDWSISINYRTP_(m)CX_(n) 300ADX_6077_F02 DG→DL mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminal PmCXnQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDLEKAATDWSISI NYRTP_(m)CX_(n) 301ADX_6077_F02 DG→DL mutant MVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDLEKAATDWSIS(G) and C-terminal P_(m)CX_(n) INYRTP_(m)CX_(n) 302ADX_6077_F02 DG→DL mutant MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDLEKAATDWSI(MG) and C-terminal P_(m)CX_(n) SINYRTP_(m)CX_(n) 303ADX_6077_F02 DG→DL mutant EVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal PmCXn (m = 1;HVTATISGLKPGVDYTITVYAVTYDLEKAATDWSISINYRTPC n = 0) 304ADX_6077_F02 DG→DL mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDLEKAATDWSISI PmCXn (m = 1; n = 0)NYRTPC 305 ADX_6077_F02 DG→DL mutantGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length w/N-terminal leader andVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDLEKAATDWSISPmCXn C-terminal modification INYRTPC (m = 1; n = 0) 306ADX_6077_F02 DG→DL mutant MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length w/N-terminal eaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDLEKAATDWSI (MG) and PmCXn C-terminalSINYRTPC modification (m = 1; n = 0) 307 ADX_6077_F02 DG→DL mutantVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDLEKAATDWSISIPmCXn (m = 1; n = 0) and His₆ tag NYRTPCHHHHHH 308ADX_6077_F02 DG→DL mutant EVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal PmCXn₁CXn₂HVTATISGLKPGVDYTITVYAVTYDLEKAATDWSISINYRTP_(m)CX_(n) ₁CX₂ 309ADX_6077_F02 DG→DL mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDLEKAATDWSISI P_(m)CX_(n1)CX₂NYRTP_(m)CX_(n1)CX₂ 310 ADX_6077_F02 DG→DL mutantGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDLEKAATDWSIS(G), and C-terminal PmCXn₁CXn₂ INYRTP_(m)CX_(n1)CX₂ 311ADX_6077_F02 DG→DL mutant MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDLEKAATDWSI (MG), and C-terminalSINYRTP_(m)CX_(n1)CX₂ PmCXn₁CXn₂ 312 ADX_6077_F02 DG→DL mutantEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal PmCXn₁CXn₂HVTATISGLKPGVDYTITVYAVTYDLEKAATDWSISINYRTPCPP (m = 1; n1 = 5; n2 = 0)PPPC 313 ADX_6077_F02 DG→DL mutantVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDLEKAATDWSISIP_(m)CX_(n1)CX_(n2) (m = 1; n1 = 5; n2 = 0) NYRTPCPPPPPC 314ADX_6077_F02 DG→DL mutant GVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDLEKAATDWSIS(G), and C-terminal PmCXn1CXh2 INYRTPCPPPPPC (m = 1; n1 = 5, n2 = 0) 315ADX_6077_F02 DG→DL mutant MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDLEKAATDWSI (MG), and C-terminalSINYRTPCPPPPPC PmCXn₁CXn₂ (m = 1; n1 = 5, n2 = 0) 316ADX_6077_F02 DG→DL mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDLEKAATDWSISIP_(m)CX_(n1)CX_(n2) (m = 1 ; n1 = 5; n2 = 0), NYRTPCPPPPPCHHHHHHand His₆ tag 317 ADX_6077_F02 DG→DV mutantEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGE coreHVTATISGLKPGVDYTITVYAVTYDVEKAATDWSISINYRT 318 ADX_6077_F02 DG→DV mutantVTYDVEKAATDWS FG loop 319 ADX_6077_F02 DG→DV mutantVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPV full-lengthQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDVEKAATDWSISI NYRT 320ADX_6077_F02 DG→DV mutant GVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDVEKAATDWSIS (G) INYRT 321ADX_6077_F02 DG→DV mutant MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDVEKAATDWSI (MG) SINYRT 322ADX_6077_F02 DG→DV mutant EVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal PmXn HVTATISGLKPGVDYTITVYAVTYDVEKAATDWSISINYRTPmXn323 ADX_6077_F02 DG→DV mutantVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminal PmXnQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDVEKAATDWSISI NYRTP_(m)X_(n) 324ADX_6077_F02 DG→DV mutant GVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDVEKAATDWSIS (G) and C-terminal PmCXnINYRTP_(m)X_(n) 325 ADX_6077_F02 DG→DV mutantMGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDVEKAATDWSI (MG) and C-terminal PmXnSINYRTP_(m)X_(n) 326 ADX_6077_F02 DG→DV mutantEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal P_(m)CX_(n)HVTATISGLKPGVDYTITVYAVTYDVEKAATDWSISINYRTP_(m)CX_(n) 327ADX_6077_F02 DG→DV mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminal PmCXnQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDVEKAATDWSISI NYRTP_(m)CX_(n) 328ADX_6077_F02 DG→DV mutant GVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDVEKAATDWSIS(G) and C-terminal P_(m)CX_(n) INYRTP_(m)CX_(n) 329ADX_6077_F02 DG→DV mutant MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDVEKAATDWSI(MG) and C-terminal P_(m)CX_(n) SINYRTP_(m)CX_(n) 330ADX_6077_F02 DG→DV mutant EVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal PmCXn (m = 1;HVTATISGLKPGVDYTITVYAVTYDVEKAATDWSISINYRTPC n = 0) 331ADX_6077_F02 DG→DV mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDVEKAATDWSISI PmCXn (m = 1; n = 0)NYRTPC 332 ADX_6077_F02 DG→DV mutantGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length w/N-terminal leader (G)VQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDVEKAATDWSIS and PmCXn C-terminalINYRTPC modification (m = 1; n = 0) 333 ADX_6077_F02 DG→DV mutantMGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length w/N-terminal eaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDVEKAATDWSI (MG) and PmCXn C-terminalSINYRTPC modification (m = 1; n = 0) 334 ADX_6077_F02 DG→DV mutantVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDVEKAATDWSISIPmCXn (m = 1; n = 0) and His₆ tag NYRTPCHHHHHH 335ADX_6077_F02 DG→DV mutant EVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal PmCXn₁CXn₂HVTATISGLKPGVDYTITVYAVTYDVEKAATDWSISINYRTP_(m)CX_(n) ₁CX₂ 336ADX_6077_F02 DG→DV mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDVEKAATDWSISI P_(m)CX_(n1)CX₂NYRTP_(m)CX_(n1)CX₂ 337 ADX_6077_F02 DG→DV mutantGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDVEKAATDWSIS(G), and C-terminal PmCXn₁CXn₂ INYRTP_(m)CX_(n1)CX₂ 338ADX_6077_F02 DG→DV mutant MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDVEKAATDWSI (MG), and C-terminalSINYRTP_(m)CX_(n1)CX₂ PmCXn₁CXn₂ 339 ADX_6077_F02 DG→DV mutantEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVQEFTVPGEcore with C-terminal PmCXn₁CXn₂HVTATISGLKPGVDYTITVYAVTYDVEKAATDWSISINYRTPCPP (m = 1; n1 = 5; n2 = 0)PPPC 340 ADX_6077_F02 DG→DV mutantVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDVEKAATDWSISIP_(m)CX_(n1)CX_(n2) (m = 1; n1 = 5; n2 = 0) NYRTPCPPPPPC 341ADX_6077_F02 DG→DV mutant GVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPfull-length with N-terminal leaderVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDVEKAATDWSIS(G), and C-terminal PmCXn₁CXn₂ INYRTPCPPPPPC (m = 1; n1 = 5, n2 = 0) 342ADX_6077_F02 DG→DV mutant MGVSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSfull-length with N-terminal leaderPVQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDVEKAATDWSI (MG), and C-terminalSINYRTPCPPPPPC PmCXn₁CXn₂ (m = 1; n1 = 5, n2 = 0) 343ADX_6077_F02 DG→DV mutant VSDVPRDLEVVAATPTSLLISWSDDYHAHRYYRITYGETGGNSPVfull-length with C-terminalQEFTVPGEHVTATISGLKPGVDYTITVYAVTYDVEKAATDWSISIP_(m)CX_(n1)CX_(n2) (m = 1; n1 = 5; n2 = 0), NYRTPCPPPPPCHHHHHHand His₆ tag 344 Human GPC3MAGTVRTACLVVAMLLSLDFPGQAQPPPPPPDATCHQVRSFFQRLQPGLKWVPETPVPGSDLQVCLPKGPTCCSRKMEEKYQLTARLNMEQLLQSASMELKFLIIQNAAVFQEAFEIVVRHAKNYTNAMFKNNYPSLTPQAFEFVGEFFTDVSLYILGSDINVDDMVNELFDSLFPVIYTQLMNPGLPDSALDINECLRGARRDLKVFGNFPKLIMTQVSKSLQVTRIFLQALNLGIEVINTTDHLKFSKDCGRMLTRMWYCSYCQGLMMVKPCGGYCNVVMQGCMAGVVEIDKYWREYILSLEELVNGMYRIYDMENVLLGLFSTIHDSIQYVQKNAGKLTTTIGKLCAHSQQRQYRSAYYPEDLFIDKKVLKVAHVEHEETLSSRRRELIQKLKSFISFYSALPGYICSHSPVAENDTLCWNGQELVERYSQKAARNGMKNQFNLHELKMKGPEPVVSQIIDKLKHINQLLRTMSMPKGRVLDKNLDEEGFESGDCGDDEDECIGGSGDGMIKVKNQLRFLAELAYDLDVDDAPGNSQQATPKDNEISTFHNLGNVHSPLKLLTSMAISVVCFFFLVH 345 Human GPC3 Adnectin bindingHQVSFF region 1 346 Human CPC3 Adnectin binding EQLLQSASM region 2 347ADX_6093_A01 core sEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPG(non-binding control) SKSTATISGLKPGVDYTITVYAVTGRGESPASSKPISINYRT 348ADX_6093_A01 full-length w/N-MGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNS leader, PmCXn C-terminalPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGESPASSKPI modificationSINYRTPCHHHHHH (m = 1; n = 0), and His6 tag 349ADX_6093_A01 full-length w/N-GVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSP leader, PmCXn C-terminalVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGESPASSKPIS modification INYRTPC(m = 1; n = 0) 350 ADX_6093_A01 full-length w/N-MGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNS leader, PmCXn C-terminalPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGESPASSKPI modificationSINYRTPCPPPPPCHHHHHH (m = 1; n = 7), and His6 tag 351 N-terminal leaderMGVSDVPRD 352 N-terminal leader GVSDVPRD 353 N-terminal leaderX_(n)SDVPRD 354 N-terminal leader X_(n)DVPRD 355 N-terminal leaderX_(n)VPRD 356 N-terminal leader X_(n)PRD 357 N-terminal leader X_(n)RD358 N-terminal leader X_(n)D 359 N-terminal leader MASTSG 360C-terminal tail EIEK 361 C-terminal tail EGSGC 362 C-terminal tailEIEKPCQ 363 C-terminal tail EIEKPSQ 364 C-terminal tail EIEKP 365C-terminal tail EIEKPS 366 C-terminal tail EIEKPC 367 C-terminal tailEIDK 368 C-terminal tail EIDKPCQ 369 C-terminal tail EIDKPSQ 3706X His tail HHHHHH 371 C-terminal tail EIEPKSS 372 C-terminal tailEIDKPC 373 C-terminal tail EIDKP 374 C-terminal tail EIDKPS 375C-terminal tail EIDKPSQLE 376 C-terminal tail EIEDEDEDEDED 377C-terminal tail EGSGS 378 C-terminal tail EIDKPCQLE 379 C-terminal tailEIDKPSQHHHHHH 380 C-terminal tail GSGCHHHHHH 381 C-terminal tailEGSGCHHHHHH 382 C-terminal tail PIDK 383 C-terminal tail PIEK 384C-terminal tail PIDKP 385 C-terminal tail PIEKP 386 C-terminal tailPIDKPS 387 C-terminal tail PIEKPS 388 C-terminal tail PIDKPC 389C-terminal tail PIEKPC 390 C-terminal tail PIDKPSQ 391 C-terminal tailPIEKPSQ 392 C-terminal tail PIDKPCQ 393 C-terminal tail PIEKPCQ 394C-terminal tail PHHHHHH 395 C-terminal tail PCHHHHHH 396 C-terminal tailPPID 397 C-terminal tail PPIE 398 C-terminal tail PPIDK 399C-terminal tail PPIEK 400 C-terminal tail PPIDKP 401 C-terminal tailPPIEKP 402 C-terminal tail PPIDKPS 403 C-terminal tail PPIEKPS 404C-terminal tail PPIDKPC 405 C-terminal tail PPIEKPC 406 C-terminal tailPPIDKPSQ 407 C-terminal tail PPIEKPSQ 408 C-terminal tail PPIDKPCQ 409C-terminal tail PPIEKPCQ 410 C-terminal tail PPHHHHHH 411C-terminal tail PPCHHHHHH 412 C-terminal tail PCGC 413 C-terminal tailPCPC 414 C-terminal tail PCGSGC 415 C-terminal tail PCPPPC 416C-terminal tail PCPPPPPC 417 C-terminal tail PCGSGSGC 418C-terminal tail PCCHHHHHH 419 C-terminal tail PCHHHHHHC 420C-terminal tail PCGCHHHHHH 421 C-terminal tail PCPCHHHHHH 422C-terminal tail PCGSGCHHHHHH 423 C-terminal tail PCPPPCHHHHHH 424C-terminal tail PCPPPPPCHHHHHH 425 C-terminal tail PCGSGSGCHHHHHH 426Exemplary linker (PSPEPPTPEP)_(n) n = 1-10 427 Exemplary linker(EEEEDE)_(n) n = 1-10 428 Linker PSTPPTPSPSTPPTPSPS 429 LinkerGSGSGSGSGSGSGS 430 Linker GGSGSGSGSGSGS 431 Linker GGSGSGSGSGSGSGSG 432Linker GSEGSEGSEGSEGSE 433 Linker GGSEGGSE 434 Linker GSGSGSGS 435Linker GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 436 LinkerGGGGSGGGGSGGGGSGGGGSGGGGS 437 Linker GGGGSGGGGSGGGGSG 438 Linker GPGPGPG439 Linker GPGPGPGPGPG 440 Linker PAPAPA 441 Linker PAPAPAPAPAPA 442Linker PAPAPAPAPAPAPAPAPA 443 Linker PSPEPPTPEP 444 LinkerPSPEPPTPEPPSPEPPTPEP 445 Linker PSPEPPTPEPPSPEPPTPEPPSPEPPTPEP 446Linker PSPEPPTPEPPSPEPPTPEPPSPEPPTPEPPSPEPPTPEP 447 Linker EEEEDE 448Linker EEEEDEEEEDE 449 Linker EEEEDEEEEDEEEEDEEEEDE 450 LinkerEEEEDEEEEDEEEEDEEEEDEEEEDEEEEDE 451 Linker RGGEEKKKEKEKEEQEERETKTP 452ADX_4578_F03 nucleotide ATGGGAGTTTCTGATGTGCCGCGCGACTTGGAAGTGGTTGCCGCCsequence encoding (SEQ ID NO:ACCCCCACCAGCCTGCTGATCTCTTGGCATCCGCCGCATCCGAAC 10)ATCGTTTCTTACCATATCTACTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGGAAGGTTCTAAATCTACTGCTAAAATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTACGCTGTTGCTCCGGAAATCGAAAAATACTACCAGATTTGGATTAATTACCGCACAGAAGGCAGCGGTTCCTAA 453 ADX_4578_H08ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATCAGCTGGTCTGGTTACGACTACGGTGACTCTTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGACGGTTCTAACACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAAGCTTACGGTAAAGGTTACACTCGTTACACTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGTAA 454 ADX_4578ATGGGAGTTTCTGATGTGCCGCGCGACTTGGAAGTGGTTGCCGCCACCCCCACCAGCCTGCTGATCTCTTGGTTCCCGGACCGTTACGTTTACTACATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGGAAGGTCATAAACAGACTGCTTACATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTACGCTATCTACTACTACCCGGACGACTTCCAGGGTTACCCGCAGCCGATTTCTATTAATTACCGCACAGAAGGCAGCGGTTCCTAA 455 ADX_4606_F06ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATCAGCTGGAACTCTGGTCATTCTGGTCAGTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTCGTTACGGTTACACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGCTCATTCTGAAGCTTCTGCTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGTAA 456 ADX_5273_C01ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATCAGCTGGTCTGACCCGTACGAAGAAGAACGATATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGCTTTCCATACTACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTTACAAACATAAATACGCTTACTACTACCCGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGTAA 457 ADX_5273_D01ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATCAGCTGGGAACCGTCTTACAAAGACGACCGATATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTTCTTTCCATCAGACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTTACGAACCGGACGAATACTACTTCTACTACCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGTAA 458 ADX_5274ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATCAGCTGGTCTGGTGACTACCATCCGCATCGATATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGGTGAACATGAAACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTTACGACGGTGAAAAAGCTGACAAATACCCGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGTAA 459 ADX_6077_F02ATGGGAGTTT CTGATGTGCC GCGCGACCTG GAAGTGGTTGCTGCCACCCC CACCAGCCTG CTGATCAGCT GGTCTGATGACTACCATGCG CATCGATATT ACCGCATCAC TTACGGCGAAACAGGAGGCA ATAGCCCTGT CCAGGAGTTC ACTGTGCCTGGTGAACATGT GACAGCTACC ATCAGCGGCC TTAAACCTGGCGTTGATTAT ACCATCACTG TGTATGCTGT CACTTACGACGGTGAAAAGG CTGCCACAGA TTGGTCAATT TCCATTAATTACCGCACACC GTGCCACCAT CACCACCACC ACTGA 460 ADX_6077_F02GGTGTT AGTGATGTTC CGCGTGATCT GGAAGTTGTT w/o His-tag and including leaderGCAGCAACCC CGACCAGCCT GCTGATTAGC TGGTCAGATG sequenceATTATCATGC CCATCGTTAT TATCGCATTA CCTATGGTGAAACCGGTGGT AATAGTCCGG TTCAAGAATT CACCGTTCCGGGTGAACATG TTACCGCAAC CATTAGCGGT CTGAAACCGGGTGTTGATTA CACCATTACC GTTTATGCAG TTACCTACGATGGTGAAAAA GCAGCAACCG ATTGGAGCAT TAGCATTAAC TATCGTACCC CGTGTTAA 461ADX_6077_F02 ATGAAAAAAATC TGGCTGGCAC TGGCAGGTCT GGTTCTGGCAw/leader sequence and PCPPPPPCTTTAGCGCTA GCGCCGGTGT TAGTGATGTT CCGCGTGATCTGGAAGTTGT TGCAGCAACC CCGACCAGCC TGCTGATTAGCTGGTCAGAT GATTATCATG CCCATCGTTA TTATCGCATTACCTATGGTG AAACCGGTGG TAATAGTCCG GTTCAAGAATTCACCGTTCC GGGTGAACAT GTTACCGCAA CCATTAGCGGTCTGAAACCG GGTGTTGATT ACACCATTAC CGTTTATGCAGTTACCTACG ATGGTGAAAA AGCAGCAACC GATTGGAGCATTAGCATTAA CTATCGTACC CCGTGTCCGC CGCCACCGCC GTGTTGATAA 462 ADX_6077_F02ATGGGAGTTT CTGATGTGCC GCGCGACCTG GAAGTGGTTG w/leader sequence andCTGCCACCCC CACCAGCCTG CTGATCAGCT GGTCTGATGA PCPPPPPCH6CTACCATGCG CATCGATATT ACCGCATCAC TTACGGCGAAACAGGAGGCA ATAGCCCTGT CCAGGAGTTC ACTGTGCCTGGTGAACATGT GACAGCTACC ATCAGCGGCC TTAAACCTGGCGTTGATTAT ACCATCACTG TGTATGCTGT CACTTACGACGGTGAAAAGG CTGCCACAGA TTGGTCAATT TCCATTAATTACCGCACACC GTGCCCGCCG CCACCGCCGT GTCACCATCA CCACCACCAC TGA 463ADX_6093_A01 full length VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGESPASSKPISI NYRT 464 linker (GS)₅₋₁₀465 linker (G₄S)₂₋₅ 466 linker (G₄S)₂G 467 linker PVGVV

1. A nucleic acid encoding polypeptide comprising a tenth fibronectintype III (¹⁰Fn3) domain comprising BC, DE and FG loops, wherein thepolypeptide binds specifically to human glypican-3 (GPC3), and wherein(a) the BC, DE and FG loops comprise SEQ ID NOs: 6, 7 and 8,respectively; (b) the BC, DE and FG loops comprise SEQ ID NOs: 19, 20and 21, respectively; (c) the BC, DE and FG loops comprise SEQ ID NOs:32, 33 and 34, respectively; (d) the BC, DE and FG loops comprise SEQ IDNOs: 45, 46 and 47, respectively; (e) the BC, DE and FG loops compriseSEQ ID NOs: 58, 59 and 60, respectively; (f) the BC, DE and FG loopscomprise SEQ ID NOs: 71, 72 and 73, respectively; (g) the BC, DE and FGloops comprise SEQ ID NOs: 84, 85 and 86, respectively; (h) the BC, DEand FG loops comprise SEQ ID NOs: 99, 100 and 101, respectively; (i) theBC, DE and FG loops comprise SEQ ID NOs: 99, 100 and 129, respectively;(j) the BC, DE and FG loops comprise SEQ ID NOs: 99, 100 and 156,respectively; (k) the BC, DE and FG loops comprise SEQ ID NOs: 99, 100and 183, respectively; (l) the BC, DE and FG loops comprise SEQ ID NOs:99, 100 and 210, respectively; (m) the BC, DE and FG loops comprise SEQID NOs: 99, 100 and 237, respectively; (n) the BC, DE and FG loopscomprise SEQ ID NOs: 99, 100 and 264, respectively; (o) the BC, DE andFG loops comprise SEQ ID NOs: 99, 100 and 291, respectively; or (p) theBC, DE and FG loops comprise SEQ ID NOs: 99, 100 and 318, respectively.2. The nucleic acid of claim 1, wherein the nucleic acid encodes apolypeptide comprising an amino acid sequence at least 90% to the aminoacid sequence of any one of SEQ ID NOs: 5, 9-18, 22-31, 35-44, 48-57,61-70, 74-83, 87-98, 102-128, 130-155, 157-182, 184-209, 211-236,238-263, 265-290, 292-317 or 319-343.
 3. The nucleic acid of claim 1,wherein the nucleic acid encodes a polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 5, 9-18,22-31, 35-44, 48-57, 61-70, 74-83, 87-98, 102-128, 130-155, 157-182,184-209, 211-236, 238-263, 265-290, 292-317 and 319-343.
 4. The nucleicacid of claim 1, wherein the nucleic acid encodes a polypeptide furthercomprising a heterologous protein.
 5. The nucleic acid of claim 4,wherein the heterologous protein comprises a polypeptide selected fromthe group consisting of a ¹⁰Fn3 domain, an Fc, Fc fragment, transferrin,serum albumin, a serum albumin binding protein, and a serumimmunoglobulin binding protein.
 6. The nucleic acid of claim 1, whereinthe C-terminus of the ¹⁰Fn3 domain comprises a moiety consisting of theamino acid sequence P_(m)X_(n), wherein P is proline, each X isindependently any amino acid, m is an integer that is at least 1 and nis 0 or an integer that is at least
 1. 7. The nucleic acid of claim 6,wherein the C-terminal moiety comprises cysteine.
 8. A pharmaceuticalcomposition comprising the nucleic acid of claim 1, and apharmaceutically acceptable carrier.
 9. A method of detecting ormeasuring the expression of glypican-3 in a sample comprising contactingthe sample with the nucleic acid of claim 1, and detecting or measuringexpression of the nucleic acid.
 10. The nucleic acid of claim 1, whereinthe nucleic acid encodes a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 98 and 102-127. 11.The nucleic acid of claim 1, wherein the nucleic acid encodes apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 263 and 265-289.
 12. A vector comprising thenucleic acid of claim
 1. 13. A cell comprising the nucleic acid ofclaim
 1. 14. A method of producing a polypeptide comprising a tenthfibronectin type III (¹⁰Fn3) domain comprising BC, DE and FG loops,wherein the polypeptide binds specifically to human glypican-3 (GPC3),the method comprising culturing the cell of claim 13 under conditionssuitable for expressing the polypeptide, and purifying the polypeptide.15. A method of detecting human glypican-3 (GPC3) in a samplecomprising: (a) contacting the sample with a polypeptide comprising afibronectin type III tenth domain (¹⁰Fn3), wherein (i) the ¹⁰Fn3 domaincomprises AB, BC, CD, DE, EF, and FG loops, (ii) the ¹⁰Fn3 has at leastone loop selected from loop BC, DE, and FG with an altered amino acidsequence relative to the sequence of the corresponding loop of the human¹⁰Fn3 domain (SEQ ID NO: 1), and (iii) the polypeptide specificallybinds to GPC3; and (b) detecting GPC3.
 16. The method of claim 15,wherein the polypeptide comprises a detectable label.
 17. The method ofclaim 16, wherein the detectable label is a radioactive agent.
 18. Themethod of claim 17, wherein the radioactive agent is selected from thegroup consisting of ¹⁸F⁶⁰Cu, ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, ¹²⁴I, ⁸⁶Y, ⁸⁹Zr, ⁶⁶Ga,⁶⁷Ga, ⁶⁸Ga, ⁴⁴Sc, ⁴⁷Sc, ¹¹C, ¹¹¹In, ^(114m)In, ¹¹⁴In, ¹²⁵I, ¹²⁴I, ¹³¹I,¹²³I, ³²Cl, ³³Cl, ³⁴C, ⁷⁴Br, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ⁷⁸Br, ⁸⁹Zr, ¹⁸⁶Re, ¹⁸⁸Re,⁸⁶Y, ⁹⁰Y, ¹⁷⁷Lu, ⁹⁹Tc, ²¹²Bi, ²¹³Bi, ²¹²Pb, ²²⁵Ac, or ¹⁵³Sm.
 19. Amethod of detecting GPC3 positive cells in a subject comprising (a)administering to the subject an imaging agent comprising a polypeptideconjugated to a detectable label, wherein the polypeptide comprises afibronectin type III tenth domain (¹⁰Fn3), wherein (i) the ¹⁰Fn3 domaincomprises AB, BC, CD, DE, EF, and FG loops, (ii) the ¹⁰Fn3 has at leastone loop selected from loop BC, DE, and FG with an altered amino acidsequence relative to the sequence of the corresponding loop of the human¹⁰Fn3 domain (SEQ ID NO: 1), and (iii) the polypeptide specificallybinds to GPC3; and (b) detecting the imaging agent, the detected imagingagent defining the location of the GPC3 positive cells in the subject.20. The method of claim 19, wherein the imaging agent is detected bypositron emission tomography.
 21. A polypeptide comprising a fibronectintype III tenth domain (¹⁰Fn3) conjugated to a radioactive agent wherein(i) the ¹⁰Fn3 domain comprises AB, BC, CD, DE, EF, and FG loops, (ii)the ¹⁰Fn3 has at least one loop selected from loop BC, DE, and FG withan altered amino acid sequence relative to the sequence of thecorresponding loop of the human ¹⁰Fn3 domain (SEQ ID NO: 1), (iii) thepolypeptide specifically binds to GPC3, and (iv) the radioactive agentis selected from the group consisting of ¹⁸F⁶⁰Cu, ⁶¹Cu, ⁶²Cu, ⁶⁴Cu,¹²⁴I, ⁸⁶Y, ⁸⁹Zr, ⁶⁶Ga, ⁶⁷Ga, ⁶⁸Ga, ⁴⁴Sc, ⁴⁷Sc, ¹¹C, ¹¹¹In, ^(114m)In,¹¹⁴In, ¹²⁵I, ¹²⁴I, ¹³¹I, ¹²³I, ³²Cl, ³³Cl, ³⁴Cl, ⁷⁴Br, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br,⁷⁸Br, ⁸⁹Zr, ¹⁸⁶Re, ¹⁸⁸Re, ⁸⁶Y, ⁹⁰Y, ¹⁷⁷Lu, ⁹⁹Tc, ²¹²Bi, ²¹³Bi, ²¹²Pb,²²⁵Ac, or ¹⁵³Sm.
 22. A pharmaceutical composition comprising thepolypeptide of claim 21.